Insulin-like growth factor II (IGF-II) binding factors

ABSTRACT

This invention relates to modified IGF-II binding domains of the Insulin-like Growth Factor 2 Receptor (IGF2R) which have enhanced binding affinity for IGF-II relative to the wild type IGF-II binding domain. Suitable IGF-II binding domains may be modified, for example, by substituting residue E1544 for a non-acidic residue. These modified domains may be useful in the sequestration of Insulin-like Growth Factor II (IGF-II), for example, in the treatment of cancer.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/GB2006/003011, filed Aug. 11, 2006, whichwas published under PCT Article 21(2) in English.

This invention relates to methods and materials for use in thesequestration of Insulin-like Growth Factor-II (IGF-II), for example, inthe treatment of cancer.

The mammalian cation-independent mannose 6-phosphate/insulin-like growthfactor II receptor (abbreviated to IGF2R) is a type I integral membraneprotein and P-type lectin, with multiple functions attributable to itswide variety of known ligands (1-3). The ˜270 kDa glycosylated proteinconsists of an N-terminal signal sequence (amino acids 1-44), 15homologous extracytoplasmic repeating domains (amino acids 45-2313), atransmembrane region (amino acids 2314-2336) and a C-terminalcytoplasmic domain (amino acids 2337-2499) (4, 5). Its 15 repeateddomains are each ˜147 amino acids in length and display significantsimilarity in amino acid sequence and disulphide distribution to eachother (16-38% identity) and with the single extra-cytoplasmic domain ofthe cation-dependent mannose 6-phosphate receptor (CD-MPR) (14-28%identity) (5). Crystal structures have now been solved for domains 1, 2,3 (6) and 11 (7) and show that each domain has a similar topologyconsisting of a flattened 9-strand β barrel, shared with the CD-MPR andavidin (8), suggesting that the 15 extracytoplasmic domains represent 15homologous structural units. The main function of the IGF2R and theCD-MPR are the delivery of newly formed acid hydrolases, of which thereare ˜50, to the lysosome through binding to their mannose 6-phosphate(M6P) labelled residues (3). IGF2R also processes a number of other M6Pand non-M6P labelled ligands. Domains 3, 9 and recently 5 (9) have beenidentified as the binding sites for the mannosylated proteins such aslatent TGF-β (10), proliferin and granzyme B and the protease cathepsin.Site-directed mutagenesis studies have since established the criticalinteracting residues within domains 3 and 9 for mannose 6-phosphate (11,12). Of the currently identified non-mannosylated ligands (IGF-II,retinoic acid, urokinase type plasminogen activator receptor andplasminogen) IGF-II has been by far the best studied with the bindingsite being localised to domain 11 (13-15).

IGF-II (7.5 kDa) is a small mitogenic peptide hormone that functionsprincipally during embryonic growth, where its activity is tightlyregulated, but is also frequently deregulated in tumours (16-18). LikeIGF-I, IGF-II exerts its mitogenic affect predominantly by signallingthrough the IGF1R, leading to tyrosine kinase activation and stimulationof both the mitogen-activated protein (MAP) kinase and PKB/AKTsignalling cascades. Downstream targets include the FOXO transcriptionfactors, GSK3β, MDM2 and mTOR leading to up regulation of pro-growth andanti-apoptotic signals (19). In mammals, tight regulation of IGF-IIactivity is achieved by high affinity binding to six IGF bindingproteins (IGFBP 1-6) and by binding to the IGF2R at the cell surface,leading to internalisation of IGFII and subsequent degradation withinthe lysosome (20-22). Previous NMR studies have established thestructure of mature IGF-II (23, 24) and site directed mutagenesis hasbeen used to identify the residues F48, R49, S50, A54, L55 as beingcritical to the interaction with IGF2R (25, 26). Although IGF-II isrelatively structurally conserved, the IGF-II binding site of IGF2R ispresent only in mammalian species, where embryonic and placental growthregulation of IGF-II by the IGF2R also involves reciprocal imprinting ofthe genes coding these proteins (27). Disruption of Igf2 in the mouseresults in reduced growth (60% of wild-type) from embryonic day 9-11(28, 29), whereas mice with disruption of Igf2r exhibit fetal overgrowthand fatal cardiac hyperplasia (30, 31). The growth and perinatallethality phenotype is rescued when Igf2 is also disrupted, suggestingthe principle critical function of IGF2R is the regulation of IGF-II(32). The specific functional interaction between IGF-II and IGF2R andits critical role in development has been highlighted more recently, asparthenogenetic embryos with maternal allele Igf2 expression can lead tonormal development of live mice with two maternal genomes, andepigenetic suppression of Igf2r may account for large offspring syndromefollowing somatic cell cloning (33, 34).

Aberrant regulation of IGF-II activity has been repeatedly implicated asa common feature of tumours in both mouse and human (35). For example,increased expression of IGF-II by loss of imprinting (LOI) has beendescribed in a plethora of tumour types including Wilms' tumour (36),colorectal carcinoma (37), rhabdomyosarcoma (38), Ewing's sarcoma (39),cervical carcinoma (40), lung carcinoma (41) and phaeochromocytoma (42).IGF2 LOI is particularly associated with increased relative risk ofdeveloping colorectal carcinoma (43, 44). IGF2R also acts as a tumoursuppressor, as loss of heterozygosity of the receptor has been detectedin a number of tumour types including liver, lung and head and necktumours (45-48). Moreover, loss of function mutations of the receptorhave been characterised (49), and over-expression of IGF2R causesdecreased growth and increased apoptosis in tumour cell models (50-53).The crystal structure of IGF2R domain 11 has been solved at 1.4 Åresolution using anomalous scattering of sulphur (7), and has beenconfirmed by others (54). Domain 11 IGF2R structure reveals twohydrophobic sites on the surface of domain 11, the first that identifiesthe putative IGF-II binding site within the cleft of the β-barrelstructure, spatially analogous to the hydrophilic sugar binding site ofthe CD-MPR, and a second that is implicated in domain-domaininteractions (7, 55). The IGF-II binding site is formed by the β-strandsA, B, C and D and the loops AB, CD and FG, with shorter loops conferringa shallower binding cavity than that of the CD-MPR. The residues Y1542,S1543, G1546 (AB loop), F1567, G1568, T1570, I1572 (CD loop), S1596,P1597, P1599 (FG loop) have been identified as being significantlysolvent exposed and therefore potentially involved in the IGF-IIinteraction (7). Previously, Linnell et al quantified the interaction ofIGF-II with IGF2R domain 10-13 expressed as a rat CD4 (domains 3 and 4)chimeric protein using surface plasmon resonance (SPR), and confirmedthe enhancing activity of domain 13 to the domain 11-IGF-II interaction(15, 56).

The present inventors have identified novel mutants of the IGF-IIbinding domain of the Insulin-like Growth Factor 2 Receptor (IGF2R)which have enhanced binding affinity for IGF-II relative to the wildtype IGF-II binding domain, but do not display any reduction inspecificity.

One aspect of the invention provides an IGF-II binding domain consistingof the amino acid sequence of residues 1511 to 1650 of human IGF2R with50 or fewer residues mutated or altered,

-   -   wherein E1544 is substituted for a non-acidic residue.

The IGF-II binding domain binds IGF-II with increased affinity relativeto the wild-type IGF-II binding domain (residues 1511 to 1650) of humanIGF2R, for example 2 fold, 3 fold, 4 fold, 5 fold, 6 fold or 7 fold ormore greater affinity. The binding of the mutant IGF-II binding domainto IGF-II may, for example, have an increased association rate (k_(on))and/or a reduced dissociation rate (k_(off)) relative to the wild typeIGF-II binding domain.

Preferably, the IGF-II binding domain shows a binding specificity whichis identical or similar to the wild-type IGF-II binding domain (residues1511 to 1650) of human IGF2R, i.e. it shows no binding or substantiallyno binding to IGF-I.

Human IGF2R has the amino acid sequence shown in SEQ ID NO: 1 anddatabase entry NP_(—)000867.1 GI: 4504611 and is encoded by thenucleotide sequence shown in SEQ ID NO: 2 and database entry(NM_(—)000876.1 GI: 4504610). The amino acid sequence of residues 1511to 1650 of human IGF2R is shown in SEQ ID NO: 3. Residue E1544 in thehuman IGF2R sequence corresponds to residue E34 of SEQ ID NO: 3. Exceptwhere otherwise stated, residue numbers set out herein refer to theposition of the residue in the human IGF2R sequence shown in SEQ ID NO:1.

A residue identified by its position in the human IGF2R sequence caneasily be identified in a truncated or variant IGF2R sequence, such asthe IGF-II binding domain sequence shown in SEQ ID NO: 3, or variantsthereof, for example, using standard sequence alignment and analysistechniques.

Other than the substitution of a non-acidic residue at E1544, a IGF-IIbinding domain described herein may have 50 or fewer amino acid residuesaltered relative to the wild-type amino acid sequence of residues 1511to 1650 of human IGF2R, preferably 45 or fewer, 40 or fewer, 30 orfewer, 10 or fewer, 5 or fewer or 3 or fewer.

An amino acid residue in the wild-type amino acid sequence may bealtered or mutated by insertion, deletion or substitution of one or moreamino acids. Such alterations may be caused by one or more of addition,insertion, deletion or substitution of one or more nucleotides in theencoding nucleic acid.

In some embodiments, residues F1567 and I1572 are not mutated, morepreferably, residues F1567, T1570 and I1572 are not mutated, and mostpreferably residues F1567, T1570, I1572, P1597 and P1599 are not mutatedi.e. a IGF-II binding domain described herein comprises F1567, T1570,I1572, P1597 and P1599 (which correspond to residues F57, T60, I62, P87and P89 of SEQ ID NO: 3, respectively). Preferably, the mutant IGF-IIdomain retains the beta-barrel structure of the wild-type domain.

In some preferred embodiments, the IGF-II binding domain may consist ofthe amino acid sequence of residues 1511 to 1650 of human IGF2R withresidue E1544 mutated by substitution for a non-acidic residue.

The mutant IGF-II binding domain may share at least 50% sequenceidentity with the wild-type amino acid sequence of residues 1511 to 1650of human IGF2R, at least 55%, at least 60%, at least 65%, at least 70%,at least about 80%, at least 90%, at least 95% or at least 98%. Thesequence may share at least 60% similarity with the wild-type sequence,at least 70% similarity, at least 80% similarity, at least 90%similarity, at least 95% similarity or at least 99% similarity.

Sequence similarity and identity are commonly defined with reference tothe algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA).GAP uses the Needleman and Wunsch algorithm to align two completesequences that maximizes the number of matches and minimizes the numberof gaps. Generally, default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of GAP may be preferred butother algorithms may be used, e.g. BLAST (which uses the method ofAltschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which usesthe method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or theSmith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147:195-197), or the TBLASTN program, of Altschul et al. (1990) supra,generally employing default parameters. In particular, the psi-Blastalgorithm may be used (Nucl. Acids Res. (1997) 25 3389-3402). Sequenceidentity and similarity may also be determined using Genomequest™software (Gene-IT, Worcester Mass. USA).

Sequence comparisons are preferably made over the full-length of therelevant sequence described herein.

Similarity allows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine.

In the mutant IGF-II binding domain, residue E1544 may be substitutedfor a non-acidic residue, for example, a residue other than E or D,preferably a residue other than E, D, N or Q, most preferably a residueother than E, D, N, P or G, i.e. the IGF-II binding domain may comprisean amino acid other than E, D, N, P or G at position 1544 (position 34in SEQ ID NO: 3).

For example, residue E1544 may be substituted for an aliphatic residuesuch as A, V, L or I, a basic residue such as K, R or H, a sulphurcontaining residue such as C or M, or a hydroxyl residue, such as S or Tmay be used. More preferably, E1544 may be substituted for a polarresidue, such as S, or a basic residue, such as K, R or H. For example,the IGF-II binding domain may comprise K, R, H or S at position 1544(position 34 in SEQ ID NO: 3). Most preferably, E1544 is substituted forR or K.

An IGF-II binding domain as described herein may comprise one or morenon-natural amino acids, modified amino acids or d-amino acids. The useof such amino acids is well-known to those of skill in the art.

A mutant IGF-II binding domain as described above may be comprisedwithin a polypeptide.

In some embodiments, the mutant IGF-II binding domain may be linked toamino acid sequences which are not linked to the wild-type IGF-IIbinding domain (domain 11) of IGF2R in nature (i.e. heterologoussequences).

In some embodiments, the polypeptide may comprise multiple IGF-IIbinding domains, including, for example, one or more mutant IGF2 bindingdomains as described herein. A polypeptide may comprise two, three, fouror more IGF-II binding domains. The presence of multiple domains mayincrease the ability of the polypeptide to bind to IGF-II. The domainsmay be identical (i.e. copies) or may be non-identical (i.e. they maydiffer at one or more amino acid residues).

The IGF-II binding domains may be directly connected without linkers ormay be linked by amino acid sequences from human IGF2R, synthetic aminoacid sequences, synthetic organic molecules or polypeptides thatmulti-merise or assemble into polymeric structures. In some embodiments,the IGF2 binding domains may be linked via biotin-streptavidin tags.

The polypeptide may further comprise one or more amino acid sequencesadditional to the one or more IGF-II binding domains. For example, theIGF-II binding polypeptide may comprise one or more additional domains.Additional domains may include domains of human IGF2R, such as domain 13(residues 1800 to 1991 of the IGF2R sequence), and domain 12 (residues1651 to 1799 of the IGF2R sequence) or domains from other polypeptides(i.e. heterologous domains) which improve the stability,pharmacokinetic, targeting, affinity, purification and productionproperties of the polypeptide, such as an immunoglobulin Fc domain,which confers improved stability/pharmacokinetic parameters inbiological fluid.

In some embodiments, the polypeptide may comprise an immunoglobulin Fcdomain. Suitable immunoglobulin Fc domains are well-known in the art andinclude the human IgG1 Fc domain. The immunoglobulin Fc domain may belocated at the N-terminal or C-terminal end of the IGF-II bindingdomain.

The immunoglobulin Fc domain and the IGF-II binding domain may beattached directly or via a linker molecule, for example a linkerpeptide.

Examples of polypeptides comprising an immunoglobulin Fc domain areshown in FIG. 8.

In preferred embodiments, the polypeptide is not comprised within afull-length IGF2R polypeptide.

In some embodiments, the polypeptide may comprise an affinity tag, whichmay, for example, be useful for purification. An affinity tag is aheterologous peptide sequence which forms one member of a specificbinding pair. Polypeptides containing the tag may be purified by thebinding of the other member of the specific binding pair to thepolypeptide, for example in an affinity column. For example, the tagsequence may form an epitope which is bound by an antibody molecule.

Suitable affinity tags include for example, glutathione-S-transferase,(GST), maltose binding domain (MBD), MRGS(H)₆, DYKDDDDK (FLAG™), T7-,S-(KETAAAKFERQHMDS), poly-Arg (R₅₋₆), poly-His (H₂₋₁₀), poly-Cys (C₄)poly-Phe (F₁₁) poly-Asp (D₅₋₁₆), Strept-tag II (WSHPQFEK), c-myc(EQKLISEEDL), Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem229, 170-9), Glu-Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Lett283, 298-302), Tag.100 (Qiagen; 12 aa tag derived from mammalian MAPkinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.)and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.). Knowntag sequences are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol.60 523-533.

In preferred embodiments, a poly-His tag such as (H)₆ or MRGS(H)₆ may beused.

The affinity tag sequence may be removed after purification, forexample, using site-specific proteases.

In some embodiments, the protein may be coupled to an appropriate signalleader peptide to direct secretion of the fusion polypeptide from cellinto the culture medium. A range of suitable signal leader peptides areknown in the art. The signal leader peptide may be heterologous to theIGF binding domain i.e. it may be a non-IGF2R signal sequence. Forexample, an α-factor secretion signal or BiP signal sequence may beemployed. Preferably, the signal peptide is removed bypost-translational processing after expression of the polypeptide.

Preferably, the polypeptide comprising or consisting of one or moreIGF-II binding domains is soluble. A soluble polypeptide does notnaturally associate with membranes after expression and does not formaggregates in aqueous solution under physiological conditions. A solublepolypeptide may, for example, lack a transmembrane domain.

Another aspect of the invention provides a nucleic acid encoding apolypeptide comprising or consisting of one or more mutant IGF-IIbinding domains as described herein.

Nucleic acid encoding a polypeptide may be comprised in a vector.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Preferably, the vector containsappropriate regulatory sequences to drive the expression of the nucleicacid in mammalian cells. A vector may also comprise sequences, such asorigins of replication and selectable markers, which allow for itsselection and replication in bacterial hosts such as E. coli.

Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate.For further details see, for example, Molecular Cloning: a LaboratoryManual: 3rd edition, Russell et al., 2001, Cold Spring Harbor LaboratoryPress. Many known techniques and protocols for manipulation of nucleicacid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, are described in detail in Current Protocols in MolecularBiology, Ausubel et al. eds. John Wiley & Sons, 1992.

A nucleic acid or vector as described herein may be introduced into ahost cell.

A range of host cells suitable for the production of recombinantpolypeptides are known in the art. Suitable host cells may includeprokaryotic cells, in particular bacteria such as E. coli, andeukaryotic cells, including mammalian cells such as CHO and CHO-derivedcell lines (Lec cells), HeLa, COS, and HEK293 cells, amphibian cellssuch as Xenopus oocytes, insect cells such as Trichoplusia ni, Sf9 andSf21 and yeast cells, such as Pichia pastoris.

Techniques for the introduction of nucleic acid into cells are wellestablished in the art and any suitable technique may be employed, inaccordance with the particular circumstances. For eukaryotic cells,suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. adenovirus, AAV,lentivirus or vaccinia. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage.

Marker genes such as antibiotic resistance or sensitivity genes may beused in identifying clones containing nucleic acid of interest, as iswell-known in the art.

The introduced nucleic acid may be on an extra-chromosomal vector withinthe cell or the nucleic acid may be integrated into the genome of thehost cell. Integration may be promoted by inclusion of sequences withinthe nucleic acid or vector which promote recombination with the genome,in accordance with standard techniques.

The introduction may be followed by expression of the nucleic acid toproduce the encoded polypeptide comprising or consisting of one or moremutant IGF2 binding domains. In some embodiments, host cells (which mayinclude cells actually transformed although more likely the cells willbe descendants of the transformed cells) may be cultured in vitro underconditions for expression of the nucleic acid, so that the encoded IGF2binding polypeptide is produced. When an inducible promoter is used,expression may require the activation of the inducible promoter.

The expressed polypeptide comprising or consisting of one or more mutantIGF-II binding domains may be isolated and/or purified, afterproduction. This may be achieved using any convenient method known inthe art. Techniques for the purification of recombinant polypeptides arewell known in the art and include, for example HPLC, FPLC or affinitychromatography. In some embodiments, purification may be performed usingan affinity tag on the polypeptide as described above.

Polypeptides comprising or consisting of one or more mutant IGF2 bindingdomains which are produced as described may be investigated further, forexample the pharmacological properties and/or activity may bedetermined. Methods and means of protein analysis are well-known in theart.

Another aspect of the invention provides a polypeptide comprising orconsisting of one or more IGF-II binding domains, a nucleic acid, or ahost cell as described herein for use in a method of treatment of thehuman or animal body by therapy. For example, the polypeptide comprisingan IGF2 binding domain, nucleic acid or host cell as described hereinmay be used in a method of treatment of cancer.

Another aspect of the invention provides the use of a polypeptidecomprising or consisting of one or more IGF-II binding domains, anucleic acid, or a host cell as described herein in the manufacture of amedicament for use in the treatment of cancer.

Cancers which may be treated as described herein include cancerscharacterised by up-regulation of IGF-II, for example colorectal cancerssuch as intestinal adenoma and colorectal carcinoma, cervical cancerssuch as cervical carcinoma, lung cancers such as lung carcinoma, kidneycancers such as Wilms' tumour, muscle cancers such as rhabdomyosarcoma,bone cancers such as Ewing's sarcoma, endocrine cancers such asphaeochromocytoma, liver cancers such as hepatocellular carcinoma, braintumours such as glioblastoma, breast cancers such as inflammatory breastcancers, upper gastrointestinal cancers such as pancreatic cancer,haematological cancers such as myeloma, soft tissue sarcomas such ashaemangiopericytoma and cancers that result in tumour relatedhypoglycaemia related to increased circulating levels of IGF2 and/orexpress the IGF-II gene at high levels.

Polypeptides as described herein may also be useful as cancer-targetingagents to deliver other anti-cancer molecules to tumours, radiolabels todetect and treat tumours, and sensitising agents that sensitise tumoursto other cancer therapies, including chemotherapy and radiotherapy.

Whilst a polypeptide comprising or consisting of one or more IGF2binding domains, a nucleic acid, or a host cell as described herein maybe administered alone, it is preferable to present it as apharmaceutical composition (e.g. formulation) which comprises thepolypeptide, nucleic acid or cell, together with one or morepharmaceutically acceptable carriers, adjuvants, excipients, diluents,fillers, buffers, stabilisers, preservatives, lubricants, or othermaterials well known to those skilled in the art and, optionally, othertherapeutic or prophylactic agents.

Methods of the invention may therefore comprise the step of formulatinga polypeptide comprising or consisting of one or more IGF2 bindingdomains, a nucleic acid, or a host cell as described herein with apharmaceutically acceptable carrier, adjuvant or excipient.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation. The precisenature of the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,or Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The pharmaceutical compositions and formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the IGF-II binding polypeptide, nucleic acid or hostcell with the carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Whether it is an IGF-II binding domain or polypeptide, nucleic acid orhost cell according to the present invention that is to be given to anindividual, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecircumstances of the individual to be treated.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.All documents mentioned in this specification are incorporated herein byreference in their entirety.

The definitions and descriptions of the features set out above apply toall aspects and embodiments which comprise those features, unlesscontext dictates otherwise.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures and tables describedbelow.

FIG. 1 shows sensorgrams obtained from varying approaches for theanalysis of IGF-II binding to domain 11 by SPR. SPR experimentsconducted using either domain 11 (a, b, c) or IGF-II (d, e, f) as theimmobilized ligand. Duplicate samples of IGF-II at 268, 134, 67, 33.5,17.8, 8.4, 4.2 and 2.1 nM injected over biotinylated CD4-11 from 293Tcells (a), amine coupled domain 11 from E. coli (b) and biotinylateddomain 11 from yeast (c). Biotinylated IGF-II immobilised to the chip,analyte injections of crude CD4-11 at 240, 120, 60, 30, 15, 7.5 and 3.8nM (d), purified 11 from E. coli at 4096, 2048, 1024, 512, 256, 128, 64,32, 16 and 8 nM (note 4096 and 2048 nM traces overlap in this example)(e) and purified 11 from yeast at 4096, 2048, 1024, 512, 256, 128, 64,32, 16 and 8 nM (f).

FIG. 2 shows the IGF-II binding site of domain 11 and the location ofmutated residues. FIG. 2( a) shows the primary amino acid sequence ofIGF2R domain 11 (amino acids 1511-1650). The loop regions that form thehydrophobic patch of the putative IGF-II binding site are boxed and thecandidate interacting residues that were mutated in this study are shownin bold. The nature of each mutated residue is indicated above(a=acidic, b=basic, h=hydrophobic and p=polar). FIG. 2( b) shows theregion of domain 11 encompassing the mutated residues (underlined in a)and the specific mutations generated.

FIG. 3 shows a diagram of a domain 11 fusion proteins (wild type orcarrying site-directed mutants) with C-terminal His₆ tag and N-terminalα-factor secretion signal. The expressed domain 11 consists of aminoacids 1511-1650 of IGF2R. Cleavage of the α-factor signal (at the siteindicated by the arrow) during secretion generates the final soluble 17kDa protein.

FIG. 4 shows sensorgrams obtained from SPR analysis of wild type andmutated IGF2R domain 11 constructs binding to IGF-II. Sensorgrams wereobtained after injections of wild type domain 11 (a) or domain 11carrying the following single-residue alterations: in loop AB, Y1542A(b), S1543A (c), E1544A (d) and K1545A (e), in loop CD, F1567A (f),Q1569A (g), T1570A (h), I1572A (i) and I1572T (j) in loop FG, S1596A (k)or in loop GH, G1619R (l). For all except (f), (i) and (j), duplicateanalyte injections were performed at concentrations of 4096, 2048, 1024,512, 256, 128, 64, 32, 16 and 8 nM. For (f), (i) and (j) analyte wasinjected at 16384, 8192, 4096, 2048, 1024, 512, 256, 128, 64 and 32 nM.Inspection of the sensorgrams show that the mutations F1567A (f), I1572A(i) and I1572T (j) all but abolish IGF-II binding, S1543A (c), Q1569A(g) and S1596A (k) have little affect and Y1542A (b) and T1570A (h) havean intermediate impact. The polymorphism G1619R (l) has an apparent wildtype interaction and the E1544A (d) mutation has a modest positiveaffect on IGF-II binding.

FIG. 5 shows sensorgrams obtained from SPR analysis of wild type domain11 and mutants at position 1544 interacting with Sensorgrams wereobtained after injections of wild type domain 11 (a) or domain 11mutants with the following substitutions at position 1544: E1544A (b),E1544V (c), E1544K (d). Duplicate analyte injections were performed atconcentrations of 4096, 2048, 1024, 512, 256, 128, 64, 32, 16 and 8 nM.The sensorgrams for E1544A (b) and E1544V (c) show a modest but visibleincrease in affinity over the wild type (a), whereas E1544K (d) clearlyshows a marked increase in affinity, with an obvious decrease indissociation rate (see Table 4).

FIG. 6 shows analysis of the binding of wild type and E1544A domain 11to IGF-II by Isothermal Titration Calorimetry FIG. 6( a) shows enthalpicheat released per second for the Blank (offset by +0.05 μcal/sec), 11(offset by −0.2 μcal/sec) and E1544A. FIG. 6( b) shows integratedbinding isotherms for the titrations of IGF-II into domain 11 (offset by−4 kcal/mol) and the E1544A mutant, after blank subtraction. Fitting ofthe experimental data to a single site model (red line) generated K_(D)values of 150±4.4 nM for domain 11 and 81±8.8 nM for E1544A. Values arethe mean±S.E. of duplicate experiments.

FIG. 7 shows a histogram showing steady-state affinities (K_(D))obtained from domain 11 constructs specifically mutated to replace theglutamate residue at position 1544 with the indicated amino acid. Theaffinity of the wildtype (WT) domain 11 has been included forcomparison. Individual columns are shaded to reflect the property of theintroduced residue (dots=hydrophobic, unshaded=polar, shaded=basic,diagonal lines=acidic). Bars=standard error of mean.

FIG. 8 shows Domain 11 of human IGF2R cloned into the expression vectorspPIC9K and pMT/BiP/V5-His B; the latter vector including a C-terminalhuman IgG1 Fc tag for dimerisation.

FIG. 9 shows that increased doses of IGF-II lead to an increase inphosphorylation of PKB in HaCaT cell lines. Samples were probed with ananti-phospho-(Ser473) PKB antibody. Equal loading was confirmed byre-probing the blot with an antibody to α-tubulin

FIG. 10 shows that increased doses of IGF-II lead to an increase inphosphorylation of PKB in immortalised mouse embryonic fibroblasts (MEF)homozygous null for Igf2 (Igf2^(−/−)) and HaCaT cell lines.

FIG. 11 shows a comparison of domain 11 constructs with two differentmutations, E1544K (which enhances IGF-II binding) and I1572A (whichinhibits IGF-II binding), either with or without an Fc tag, in theirability to block the actions of IGF-II in Igf2^(−/−) MEFs.

FIG. 12 shows a comparison of Fc homodimers proteins in their ability toblock the actions of IGF-II in Igf2^(−/−) MEFs. The 11^(E1544K)-Fcconstruct significantly inhibited IGF-II-stimulated proliferation(*p=0.024, n=3 experiments with triplicate samples, ±SEM) compared with11-Fc even though the affinity for IGF-II is the same order ofmagnitude.

FIG. 13 shows that 11^(E1544K)-Fc decreases IGF-II-stimulatedproliferation of HaCaT cells in a dose-dependent manner, as determinedby ³H-thymidine incorporation.

FIG. 14 shows that 11^(E1544K)-Fc blocks proliferation and signaling inan IGF-II dependent manner in both HaCaT and immortalised Igf2^(−/−)MEFs, as determined by 3H-thymidine incorporation.

Table 1 shows the oligonucleotide primers which were used to generatesite-directed mutants. The sequence of the forward and reverseoligonucleotide primer pairs used for site-directed mutagenesis togenerate domain 11 constructs encoding single residue changes. Bold typeindicates the altered codon and the mutated bases are shown in lowercase. (NM_(—)000876.1 GI: 4504610 Human IGF2R).

Table 2 shows the SPR kinetic data for the interaction of IGF2 withdomain 11 purified from either E. coli or Pichia pastoris. Values arethe mean±S.E.M. of three separate experiments.

Table 3 shows kinetic data obtained from SPR analysis of domain 11mutants interacting with IGF2. Relative affinity was calculated bydividing steady-state values for KD by that obtained from the wild typedomain 11 receptor. Values are the mean±S.E.M. of three separateexperiments.

Table 4 shows kinetic data obtained from SPR analysis of domain 11mutated at position 1544 interacting with IGF2. Values are themean±S.E.M. of three separate experiments.

Table 5 shows kinetic data obtained from SPR analysis of domain 11 inwhich E1544 was replaced with all possible amino acid residues.

Table 6 shows a comparison of IGF2 dissociation affinity constants(K_(D)) of selected domain 11 constructs determined using either Biacoreor ITC

Table 7 shows the calculated masses, isoelectric points, and molarextinction coefficients of the IGF2R domain 11 proteins describedherein.

Table 8 shows kinetic and affinity constants for IGF2 binding to IGF2Rdomain 11 proteins described herein.

EXPERIMENTS

Methods

Construction of Plasmids for Expression of IGF2R Domain 11 andSite-Directed Mutants

Domain 11 of IGF2R was amplified by PCR from the pEFBOS plasmidcontaining domains 10-13 previously described by Linnell et al (15) andderived from ATCC (J03528), using primers 11EF(5′-AAAAGAATTCAACGAGCATGATGA-3′) (SEQ ID NO: 4) and 11AR(5′-AAAACCTAGGGGTCGCTTGCTCGCAGGC-3′) (SEQ ID NO: 5) incorporating EcoRIand AvrII restriction sites respectively. The product was blunt-endligated into the pCR-Blunt vector (Invitrogen) to generate pCR-11.Mutant constructs encoding single-residue changes were generated usingthe QuikChange II Site-directed Mutagenesis kit (Stratagene) accordingto the manufacturer's protocols, using pCR-11 as template and primerpairs as shown in Table 1. The desired mutations were confirmed by DNAbidirectional sequencing performed by The Sequencing Service (School ofLife Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) usingApplied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystemsmodel 3730 automated capillary DNA sequencer.

Wild type and mutant constructs were excised with EcoRI and AvrII forcloning into the Pichia pastoris expression vector pPIC-HIS, generatingpPIC-11 (either wild type or with the indicated mutation). The pPIC-HISvector used in this study was derived from Invitrogen's pPIC-9Kexpression plasmid in which high-level expression is driven from themethanol inducible AOX1 promoter and the expressed protein is targetedfor secretion into the growth media by fusion to the Saccharomycescerevisiae mating pheromone α-factor. This signal sequence isefficiently cleaved during secretion resulting in native protein. ThepPIC-HIS vector for expression of wild type and mutant domain 11constructs was generated by altering pPIC-9K by the insertion of adouble-stranded linker, formed by annealing the followingoligonucleotides HISF (5′-CTAGGCATCATCACCATCACCATTAAG-3′) (SEQ ID NO: 6)and HISR (5′-CTAGCTTAATGGTGATGGTGATGATGC-3′) (SEQ ID NO: 7), into theAvrII restriction site. The linker was designed such that cloning of thedomain 11 constructs into the EcoRI and resultant AvrII site wouldresult in an in-frame fusion with a C-terminal His6 tag for detectionand purification. The pPIC-Bio vector for the expression in Pichia ofdomain 11 with a biotinylation motif was similarly generated byinsertion of a double stranded linker, formed by annealing the followingoligonucleotides;BIOF(5′-CTAGGGGTCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAG) (SEQ ID NO:8) and, BIOR(5′-CTAGCTTCGTGCCATTCGATTITCTGAGCCTCGAAGATGTCGTTCAGACCC)(SEQ ID NO: 9); into the AvrII restriction site of pPIK-9K. The linkerencodes the Avitag sequence for efficient labelling with biotin, usingthe BirA enzyme (Avidity, Denver, USA). Subsequent cloning of domain 11into the EcoRI and AvrII restriction sites resulted in an in-frameC-terminal fusion. For IGF2R domain 11 protein expressed in bacteria,domain 11 was subcloned into pET-15 vector, expressed, protein purifiedand refolded as described (7).

Transformation and Expression of Domain 11 and Mutant Constructs in 293TCells

Transfections and expression of CD4-IGF2R domain 11 constructs(wild-type and I1572T mutants) were as described (15).

Transformation and Expression of Domain 11 and Mutant Constructs inPichia Pastoris

Targeted integration of the expression constructs into the his4 locus ofthe P. pastoris genome was achieved by linearising approximately 5 μg ofthe relevant vector within the HIS4 gene using SalI, before transforminginto the histidine auxothrophic P. pastoris strain GS115 (Invitrogen) byelectroporation, following Invitrogen's instructions. Briefly, anovernight 5 ml yeast culture was grown in YPD (1% yeast extract, 2%peptone 2% glucose) to A600˜2.0, this was diluted to A600˜0.1 in 50 mlof YPD and grown until A600 reached ˜1.5 (4-5 hours). After washing,cells were resuspended by adding 200 μl of 1M ice-cold sorbitol. 80 μlof the cell mixture was then added to the transforming DNA in a 0.2 cmGene Pulser cuvette (BioRad) and electroporated at 1500 kV, 25 μF, 400Ω.1 ml of 1M ice-cold sorbitol was immediately added to the mixture beforespreading on MD (1.34% YNB, 4×10−5% biotin, 2% dextrose, 2% agar) platesto select for histidine prototrophs.

For expression, each strain was grown at 30° C., 250 r.p.m. in 250 mlconical flasks containing 50 ml of BMGY (1% yeast extract, 2% peptone,100 mM potassium phosphate pH6.0, 1.34% YNB, 4×10−5% biotin, 1%glycerol), inoculated from a 5 ml overnight starter culture, for 24hours (A600˜6.0) IGF2R domain 11 mutagenesis and IGF-II binding kineticsbefore the cells were harvested by centrifugation and induced to expressby being transferred to 50 ml of BMMY (1% yeast extract, 2% peptone, 100mM potassium phosphate pH6.0, 1.34% YNB, 4×10−5% biotin, 0.5% methanol).Cultures were supplemented with methanol to a final volume of 1% after afurther 24 and 48 hours, after which the cells were removed bycentrifugation and the supernatant was retained. Supernatants weresubjected to SDS-PAGE and analysed both by staining with coomassie andby western blot. For proteins carrying the His6 tag, blots were probedusing an Anti-His6 mouse monoclonal antibody conjugated to peroxidase(Roche Diagnostics) and visualised using ECL (Amersham Biosciences).

Purification of Yeast Expressed Proteins

50 ml of yeast supernatant was passed through a 0.22 μm filter,concentrated in an Amicon Ultra centrifugal concentrator (10 kDa) andpurified using a His.Bind Quick 300 Cartridge (Novagen) following themanufacturer's protocols, followed buffer exchange with (10 mM HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20).

Concentrations were determined by measuring absorbance at 280 nm andapplying extinction coefficients as calculated by ProtParam(http://us.expasy.org/tools/protparam.html).

Circular Dichroism Spectroscopy

CD spectra were obtained from 0.1 mg/ml solution of protein in 10 mMsodium phosphate buffer pH 7.4 using a Jobin-Yvon CD6spectropolarimeter, over a wavelength range from 190-250 nm across a 1mm path length. UV spectra of 10 measurements were averaged andcorrected for the solvent CD signal.

Analytical Gel Filtration

Analytical gel filtration was carried out on a Superdex 75 HR 26/60column (Amersham Biosciences) equilibrated with HBS-E buffer, linked toan AKTA Purifier system (Amersham Biosciences). The column wascalibrated with BSA (66 kDa), carbonic anhydrase (29 kDa), andcytochrome c (12.5 kDa). Purified protein was loaded at ˜5 mg/ml. Themolecular weight of domain 11 was determined from a plot of the Ve/Vovs. log (MW) of the standards, where Ve is the elution volume of theprotein and Vo is the void volume. The void volume for the column wasdetermined by the elution of blue dextran (2000 kDa).

Analytical Ultracentrifugation

Sedimentation equilibrium experiments were performed in a Beckman XL-Aanalytical ultracentrifuge (Beckman, Palo Alto, Calif., USA) asdescribed previously (79). Data were fitted by non-linear least-squaresanalysis using ORIGIN software (80), to a single value for M_(w,app)using the partial specific volume calculated by SEDNTERP(80). Fits wereevaluated on the basis of random variation in the residuals, where theresiduals represent the difference between each data point and thecorresponding theoretical point on the fitted curve.

Surface Plasmon Resonance Analysis

Kinetic analysis by Surface Plasmon Resonance (SPR) was conducted on aBIAcore 3000 biosensor (BIAcore, Uppsala, Sweden) at 25° C. in HBS-EP(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) ata flow rate of 40 μl/min. After preconditioning the sensor chip with 3×1min injections of 1 M NaCl, 50 mM NaOH, approximately 50 Response Units(RU) of recombinant human Biotinyl-IGF-I or Biotinyl-IGF-II (GroPep Ltd,Adelaide, Australia) was immobilised on a Sensor Chip SA by affinitycapture to streptavidin. Kinetic experiments consisted of a two minuteinjection of analyte followed by a 100 s dissociation phase in runningbuffer, after which the binding surface was regenerated with a 2 minuteinjection of 2 M MgCl₂. A blank flow cell was used for in-line referencesubtraction of changes due to differences in refractive index of runningbuffer versus sample and a buffer-only injection was used to subtractinstrument noise and drift. Injections were performed in duplicate foreach concentration and in a randomised order. Kinetic parameters weredetermined by global fitting of sensorgrams to a two-state(conformational change) binding model using BIAevaluation softwareversion 4.0.1. In all cases the minor component made an insignificantcontribution to the overall affinity and as such only the kineticparameters of the major binding component were used. For eachinteraction the dissociation affinity constant (K_(D)) was alsocalculated by fitting of the response of each concentration atequilibrium to a steady-state affinity model using BIAevaluation. Inorder to investigate the influence of ionic strength, kineticexperiments were conducted in HBS-EP running buffers containing thedesired NaCl concentration. In each case, analyte samples were made upin the appropriate running buffer prior to injection.

Data for thermodynamic analysis was obtained on a BIAcore T100 byconducting kinetic experiments at temperatures of 20° C., 25° C., 30°C., 37° C. and 42° C. in HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.05% surfactant P20). Kinetic analysis at each temperature wasconducted as above, using ˜50 RU of immobilised biotinylated IGF2 on aSA sensor chip surface. Duplicate injections of analyte were performedat 512, 256, 128, 64, 32, 16, and 8 nM after each of which the surfacewas regenerated using a 2 min injection of 2 M MgCl. A blank flow celland buffer blank injections were used for reference subtraction. Thedata were analysed using the BIAcore T100 evaluation wizard. Sensorgramsgenerated at each temperature were fit to a two-state reaction model.The derived kinetic and affinity parameters of the major component werefit to the linear forms of the van't Hoff and Eyring equations to obtainvalues for ΔH°, ΔS°, ΔH°^(‡) and ΔS°^(‡).

Kinetic binding experiments for Fc-tagged IGF2R domain 11 proteins werecarried out at 25° C. at a 75 μl/min flow rate in HBS-EP binding buffer.For kinetic assays, six concentrations of Fc-tagged IGF2R domain 11protein were prepared by performing two-fold serial dilutions (inHBS-EP) ranging from 2.464 nM to 0.077 nM. A buffer control and areference flow cell were included. Analytes were injected over theligand surface for 3 min, following which the analyte solutions werereplaced by HBS-EP buffer for 1 hour. Regeneration of the sensor chipfor subsequent injections was accomplished by a 60 μl injection of 2MMgCl₂. All experiments were repeated in triplicate. Data transformationand overlay plots were prepared with BIAevaluation software version4.0.1. The reference flow cell data were subtracted and the regenerationand air spikes deleted. Curves were x and y-transformed and the buffercontrol subtracted. Data was fitted simultaneously and as muchassociation and dissociation data included as possible. Injection startand stop points were set precisely and the data fit using the bivalentanalyte model for curve fitting without bulk refractive index change.Mass transfer control experiments were performed by injecting 0.616 nM11^(E1544K)-Fc protein at five flow rates: 5, 20, 40, 60 and 75 μl/min.Binding curves were compared for consistency.

Isothermal Titration Calorimetry

Calorimetric measurements were performed using a VP-ITC microcalorimeter(MicroCal). All solutions were thoroughly degassed before use bystirring under vacuum. Titrations were performed at 25° C. and consistedof 17×15 μl injections (after an initial 7.5 μl injection) of 40 μMIGF-II in HBS-E buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA) ata rate of 1 injection every 3 mins. The sample cell contained eitherwild type domain 11 (7 uM) or the E1544A mutant (8 μM), also in HBS-Ebuffer. To correct for the heat effects of dilution and mixing, reactionheats obtained from a control experiment, in which IGF2 was injectedinto HBS-E, were subtracted. Calorimetric data were analysed usingMicroCal ORIGIN software (version 5.0) supplied with the instrument.Thermodynamic parameters were derived by fitting the binding isothermsto a single-site binding model. Values for dissociation affinityconstants (KD) were determined from duplicate experiments.

Construction, of Fc Tagged IGF-II Ligand Trap Expression Vectors

Domain 11 of M6P/IGF2R cDNA was PCR amplified from plasmid pEFBOS_1-15(17) using Pwo polymerase (Roche) and the BglII site containing forwardprimer Bg1-11-forward (5′-AAAAAAAAAGATCTCCCATGAAGAGCAACGAGCATGAT-3′)(SEQ ID NO: 10) and the AgeI site containing reverse primerAge-11-reverse (5′-AAAAACCGGTGCAGGCCAGCGGCGTGTG-3′) (SEQ ID NO: 11). ThePCR product was desalted using Microcon YM-100 filters (Millipore) anddigested with the restriction enzymes BglII and Agel. The digested PCRproduct was gel purified using Geneclean (Q-BlOgene, Cambridge, UK) andcloned into BglII and AgeI double-digested, gel purified Drosophilaexpression vector pMT/BiPN5-His B, to create pDes11. This construct wasthen C-terminally tagged with the human IgG1 Fc domain as a dimerisationmotif. To achieve this, Fc domain cDNA was PCR amplified from IMAGEclone 4851063 (ATCC-6878978) using Pwo polymerase and the AgeI sitecontaining primers Age-Fc-forward(5′-AAAAACCGGTGAGCCCAAATCTTCTGACAAAACTC-3′) (SEQ ID NO: 12) andAge-Fc-reverse (5′-AAAAACCGGTTTTACCCGGAGACAGGGAGAGG-3′) (SEQ ID NO: 13)according to rationale described previously (82). The PCR product wascleaned with a Microcon YM-100, digested with AgeI, purified byGeneclean and cloned into AgeI digested and CIAP dephosphorylated pDes11to create pDes11-Fc. Orientation of the cloned Fc gene was determined byPCR using the Bg1-11-forward and Age-Fc-reverse primers. To facilitatethe future cloning of 11-Fc into other expression vectors, the Age1 sitelinking domain 11 to the Fc tag was removed using the Stratagene ExSitemutagenesis kit and the 5′ phosphorylated oligos Fc ExSite forward(5′-GAGCCCAAATCTTCTGACAAAACTCACAC-3′) (SEQ ID NO: 14) and 11 ExSitereverse (5′-TTCGGTCGCTTGCTCGCAGG-3′) (SEQ ID NO: 15). Site directed nulland enhanced mutant versions of domain 11 were made using the oligos andprotocols described (75), namely 11^(E1544K) and 11^(11572A). Fc-taggeddomain 11^(11572A) (null mutant) and Fc-tagged domain 11^(E1544K)(enhanced mutant) expression vectors were thus generated, and were namedpDes11^(11572A)-FC and pDes11^(E1544K)-FC respectively. All constructswere verified by DNA sequencing, performed by the University of DundeeSequencing Service. The proteins produced from these vectors were named11-Fc, 11^(11572A)-Fc and 11^(E1544K)-Fc.

Production of Fc-Tagged IGF-II Ligand Trap Proteins in DrosophilaMelanogaster Cells

D.Mel-2 serum-free adapted Drosophila melanogaster Schneider 2 cellswere maintained in 5 ml of Drosophila-SFM (serum-free media)supplemented with 16.5 mM L-glutamine, at 28° C. in T-10 tissue cultureflasks. The cells were seeded at 1×10⁵ cells/ml and split when theyreached 1×10⁷ cells/ml. Cells were transfected at 70% confluency in aT-175 flask by complexing 48 μg of pDes11-Fc plasmid DNA with 96 μlTransFectin reagent (BioRad) according to the manufacturer'sinstructions. Transfected cells were cultured in 30 ml of medium total.Twenty four hours post transfection, 30 μl of 500 mM filter sterilisedcopper sulphate was added to induce transgene expression. The cells weremaintained in culture for a further 72 hours to secrete folded proteinexpressed from the transgene. Sodium phosphate was then added to thecell supernatant to a final concentration of 20 mM and the pH adjustedto 7. The supernatant was filter-sterilised and Fc-tagged proteinaffinity purified with 3 ml ProteinA Fast Flow Sepharose (AmershamBiosciences) in a column, according to the manufacturer's instructions.Bound protein was washed with 5 column volumes of 20 mM sodiumphosphate, pH 7, and eluted in 5 column volumes of 0.1 M sodium citrate,pH 3.5. The eluted protein was rapidly neutralised with 0.1 volumes of 1M Tris, pH 9, and the pH adjusted to 7.4. Buffer exchange into PBS andprotein concentration was performed using 30 kDa MWCO Amicon Ultra-15filters. The column was regenerated with 0.1 M sodium citrate, pH 3, andstored in 20% ethanol.

Measurement of Protein Size and Purity

The absorbance at 280 nm of purified protein was measured and proteinconcentration calculated using the extinction coefficient and massdetermined by the ProtParam tool available on the ExPASy.org website.Fc-tagged proteins expressed in Drosophila cells are N-glycosylated withtwo 982.9 Da molecules of Man₃GlcNAc₂ (39), and so this was included inthe mass calculation. Purified protein was filter sterilised and storedat 4° C. in aliquots. Protein samples were denatured and electrophoresedby SDS-PAGE and visualised by Coomassie staining against Precisionmarkers (BioRad).

Analytical Gel Filtration (FPLC)

To further purify proteins and measure their native mass, FPLC was usedas described previously (37). For the Fc-tagged proteins, FPLCanalytical gel filtration was carried out on a Superdex 200 HR 10/30column (Amersham Biosciences) equilibrated with 10 mM HEPES (pH 7.4),150 mM NaCl, 3 mM EDTA and linked to an ÄKTA Purifier system (AmershamBiosciences). The column was calibrated with amylase (200 kDa), alcoholdehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), andcytochrome C (12.5 kDa). Purified protein was loaded at ˜5 mg/ml. Themolecular weights of the proteins were determined from a plot of theVe/Vo vs. log(MW) of the standards, where Ve is the elution volume ofthe protein and Vo is the void volume. The void volume for the columnwas determined by the elution of blue dextran (2000 kDa).

Cell Culture

HaCaT human keratinocytes were grown in DMEM/F-12 (1:1) supplementedwith 10% foetal bovine serum (FBS), 0.5 μg/ml hydrocortisone, 50 IU/mlpenicillin, 5 μg/ml streptomycin and 1 mM L-glutamine. ImmortalisedIgf2^(−/−) mouse embryonic fibroblasts (MEFs) were derived by us fromE14 embryos (Igf2^(−/−)) using established procedures. Briefly, theembryo was washed in PBS, the head and liver were removed and the embryodesegregated with forceps and cells were allowed to grow out to form amonolayer. These cells were immortalised using the 3T3 method (76).Cells were grown in DMEM supplemented with 10% FBS, 50 IU/ml penicillin,5 μg/ml streptomycin and 1 mM L-glutamine. All cells were maintained at37° C. in 5% carbon dioxide with humidity.

Western Blot Signaling Analysis

24 hours after seeding onto 6-well plates (1×10⁵ cells per well), cellswere serum starved overnight prior to stimulation. IGFs and domain 11constructs were pre-incubated as appropriate in serum-free media at roomtemperature for 10 min before placing on the cells for a further 10 min.After stimulation cells were washed twice with ice-cold PBS andimmediately scraped into 100 μl lysis buffer (50 mM Tris-HCl, pH 7.5, 1%NP40, 1 mM EDTA, 120 mM NaCl, 40 mM β-glycerophosphate, 1 mMbenzamidine, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 1 μg/ml leupeptin, 1μg/ml antipain, 1 μg/ml pepstatin). Insoluble material was removed bycentrifugation and proteins were separated under reducing conditions on12% SDS-PAGE and transferred to PVDF membrane (Millipore) beforedetection with anti-phospho-(Ser473) PKB or anti-phospho-p44/42 MAPK(Thr202/Tyr204) antibodies following the manufacturer's instructions.Proteins were visualised using enhanced chemiluminescence (ECL)reagents. Equal protein loading was verified using an anti-α-tubulinantibody (Sigma).

³H-Thymidine Incorporation Assay

Cells were seeded onto 24-well plates (HaCaT, 1×10⁴ cells per well, MEFs3×10⁴ cells per well) in growth media. After 24 hours HaCaT cells wereserum starved for 24 hrs and then treated with appropriate IGF anddomain 11 constructs pre-incubated at room temperature in 500 μl ofserum-free medium for 10 min. After a further 24 hrs 1 μCi³H-thymidine/well was added and the cells incubated for 1 hour. Mediawas removed and cells washed twice with PBS before fixation in 500 μl 5%TCA for 20 min at 4° C. followed by extraction in 400 μl 0.1 M NaOH at4° C. for 1 hour. MEFs were serum starved overnight before stimulationwith appropriate IGF and domain 11 constructs pre-incubated in 500 μl ofserum free media at room temperature for 10 min. 1 μCi ³H-thymidine perwell was added with the IGFs and cells incubated for 24 hrs beforefixation in 5% TCA and extraction with 0.1 M NaOH. Incorporation of³H-thymidine was analysed by scintillation counting.

Statistical Analysis

Data were analysed with the student's t-test (MINITAB version 14software, Minitab Inc., Pennsylvania, USA).

Results

Optimisation of SPR Kinetic Analysis

A chimeric protein of IGF2R domain 11 fused to rat CD4 domains 3 and 4and carrying a biotinylation motif was generated in an analogous fashionto the multidomain proteins used previously by Linnell et al (15). SPRanalysis was conducted using a BIAcore 3000 with biotinylated CD4-11 asthe immobilised ligand, captured via streptavidin to the sensor chipsurface. 675 RU of CD4-11 (45 kDa) was immobilised to achieve atheoretical Rmax of ˜110 RU. A non-chimeric biotinylated CD4 wasimmobilised to an upstream flow cell and used for in-line referencesubtraction. Using IGF-II as the analyte, experiments were performedusing duplicate analyte injections of 268, 134, 67, 33.5, 16.8, 8.4, 4.2and 2.1 nM. The resulting sensorgram profiles indicated a complexinteraction mechanism that did not fit a 1:1 langmuir model (FIG. 1 a).The failure of even the highest concentration to reach equilibrium overthe course of the injection and failure of curves to return to thestarting baseline suggests a biphasic interaction with one componentthat dissociated rapidly, and another that appeared to remain bound. Theexperiment was repeated using a CD4-11 construct carrying an I1572Tmutation as the in-line reference. This mutation had previously beenidentified as abolishing IGF-2 binding (13, 15) and as such shouldprovide a suitable reference for the subtraction of any non-specificbinding. However, no non-specific binding to the control surface wasdetected, indicating that the apparent complex interaction was not dueto non-specific binding but dependent upon an intact IGF-II bindingsite. Repeated injections of IGF-II without either regeneration orextended IGF-II injections were able to achieve response levels inexcess of the theoretical R_(max), providing indication that theobserved sensorgram shape was not the product of a 1:1 interaction ofIGF-II and the domain 11 binding pocket. Experimental conditions weremodified to minimise this effect. This included decreasing theimmobilisation level (down to 20 RU), blocking the remainingstreptavidin binding sites with free biotin (0.5 μM) or biotinylated CD4(30 μg/ml), blocking non-specific sites by preinjecting with bovineserum albumin (Fraction V BSA, 1 mg/ml), including BSA (1 mg/ml) as acarrier during the injections, using running buffers at either pH 7, 6,or 5 or performing the experiments at either 37° C. or 20° C. However,such conditions did not prevent the apparent concentration dependentupward shift in baseline following dissociation and, as the sensorgramsdid not fit a 1:1 interaction model, it was not possible to obtainkinetic data for the interaction using this approach.

A number of alternative strategies were attempted using either IGF-II ordomain 11 as the immobilised binding partner. For each experiment theligand was immobilised to achieve a theoretical Rmax of ˜100 RU. Wefirst expressed a native domain 11 protein in E. coli, that was thenrefolded and purified before amine coupling to a CM5 sensor chip.Duplicate injections of 268, 134, 67, 33.5, 17.8, 8.4, 4.2 and 2.1 nMIGF-II were performed. The sensorgram (FIG. 1 b) showed that theimmobilised domain 11 surface was of a very low activity providingindication that very little of the immobilised protein was functional.This result may be either due to inactivation of the protein as a resultof amine coupling, or covalent bonds formed between the chipcarboxy-methyl dextran and lysine residues of the domain 11 IGF-IIbinding site. As an alternative, a domain 11 protein (amino acids1511-1650) with a C-terminal biotinylation motif was then expressed inPichia pastoris (59). The soluble secreted protein (0.5-2 mg/L ofculture supernatant) was biotinylated in the culture supernatant (BirA)and immobilised to the sensor chip surface by affinity capture of biotinto streptavidin (10-15M).

Duplicate injections of 268-2.1 nM IGF-II were performed. The resultingsensorgrams again showed the apparent complex response (FIG. 1 c),providing indication that the observed effect was dependent on theinteraction of immobilised domain 11 with IGF-II in the flow buffer,rather than an artefact associated with the fusion of domain 11 to CD4.With biotinylated IGF-II immobilised to the cell surface, duplicateinjections of nonbiotinylated CD4-11 were then performed at 240, 120,60, 30, 15, 7.5 and 3.8 nM. The resulting sensorgrams (FIG. 1 d) alsoappeared to show a similar complex interaction.

Repeating the approach using immobilised biotinylated IGF-II, andpurified soluble domain 11, either produced from E. coli or Pichiapastoris, as the analyte at concentrations of 4096, 2048, 1024, 512,256, 128, 64, 32, 16 and 8 nM, the sensorgrams (FIGS. 1 e and f,respectively) showed that, under these experimental conditions, theinteraction conforms to a standard 1:1 model, with each curve returningto the original baseline within 2-3 minutes. Kinetic parameters wereassigned by global fitting the observed sensorgrams to a 1:1 langmuirmodel using BIAevaluation software version 4.0.1 (Table 2). Asequilibrium was achieved over the course of the injection for eachconcentration, the dissociation affinity constant (KD) was alsocalculated at steady-state binding. The interaction of IGF2 with domain11, whether obtained from E. coli or Pichia pastoris, had similarkinetic association and dissociation rates and a KD of ˜100 nM, whetherdetermined from the kinetic constants or steady state binding (Table 2).Moreover, calculation of the stoichiometry from steady state Rmaxindicated that at least 80% of the soluble domain 11 expressed in eithersystem bound IGF-II. As yeast culture supernatant provided a simple,rapid and economic method for the production of correctly folded andsoluble domain 11, this method was chosen for the expression of allfurther constructs. SPR analysis was also performed in all subsequentexperiments with biotinylated IGF-II immobilised to a streptavidincoated chip surface and domain 11 was used as the analyte.

Generation of Site-Directed Mutants within IGF2R Domain 11

Site-directed mutagenesis of pCR-11 followed by sub-cloning intopPIC-HIS was used to generate mutated domain 11 constructs, with aC-terminal His6 tag, for secreted expression in Pichia pastoris.Selected mutagenesis of individual amino acids was used to assess theircontribution to IGF-II binding (See Table 1 for sequence of mutagenicprimers). Of the solvent exposed amino acid side chains identified byBrown et al (7) as being potentially involved in IGF-II binding Y1542,S1543 (AB loop), F1567, T1570, I1572 (CD loop) and S1596 (FG loop) wereinitially mutated to alanine (FIG. 2). The role of residues G1546,G1568, P1597 and P1599 was not initially assessed as mutation of eitherglycine or proline would be predicted to significantly alter theconformation of the binding site. E1544 and K1545 (AB loop) were alsomutated. I1572 was also mutated to threonine as this amino acidsubstitution had previously been characterised as abolishing IGF-IIbinding, and acted as a positive control (13, 15). A glycine to argininesubstitution at position 1619 (GH loop) has been identified as a commonhuman polymorphism within domain 11 of the IGF2R (FIG. 2) (60). In orderto assess the functional consequence of this polymorphism a furtherconstruct bearing the G1619R substitution was also generated.

Expression and Purification of Domain 11 Constructs in Pichia Pastoris

Domain 11 and the site-directed mutant forms were expressed as secretedsoluble proteins in the yeast Pichia pastoris using the pPIC-HISexpression plasmid (FIG. 3). Following induction, analysis of each yeastsupernatant by immunoblot, probed with an anti-His6 polyclonal antibody,showed the presence of the expected 17 kDa product carrying the His6epitope tag. Coomassie staining of SDS-PAGE gels also only detected the17 kDa product, indicating that the expressed protein was the onlysignificant protein present in the yeast supernatants. Proteins werepurified by metal chelation affinity chromatography, with typical yieldsof approximately 5 mg of purified protein per litre of supernatant.

Evaluation of Site-Directed IGF2R Domain 11 Mutants by SPR

Following purification, the binding of each mutant construct to IGF2 wasassessed by SPR and compared to the wild type domain 11. Sensorgramswere generated using biotinylated IGF2 immobilised to the cell surfaceand mutant domain 11 proteins as the free analyte (FIG. 4). For allconstructs, except F1567A, I1572A and I1572T, analyte was injected induplicate at concentrations of 4096, 2048, 1024, 512, 256, 128, 64, 32,16 and 8 nM. A higher concentration range of 16384, 8192, 4096, 2048,1024, 512, 256, 128, 64 and 32 nM was used for the analyte injections ofF1567A, I1572A and I1572T, as pilot experiments had shown these to havea significantly reduced IGF-II affinity compared to the other mutants.As before, kinetic parameters and steady-state affinity constants weredetermined from the sensorgrams (Table 3).

Despite the increased analyte concentration range, neither kinetic oraffinity data could be obtained for F1567A, I1572A or I1572T; thesensorgrams showed binding to be almost completely abolished for each ofthese mutations (FIGS. 4 f, i and j respectively). Interestingly, thesensorgrams show an observable increase in binding to the I1572T mutantover that of I1752A, although insufficient to be accurately quantified(Table 3). Nevertheless, this confirms previous studies that havedemonstrated I1572 as being essential to IGF-II binding (13, 15). Here,F1567 is identified herein as a second critical hydrophobic amino acidessential for IGF2R binding to IGF2 when mutated alone. All othermutants exhibited levels of IGF2 binding that were quantifiable by SPR,permitting assignment of kinetic parameters and steady-state affinity(Table 3). In all cases, the KD obtained from steady-state affinityanalysis agreed closely with that calculated from the kinetic data. Ofthe remaining mutants, the T1570A mutation had the next greatestreduction in affinity (KD˜900 nM) when compared to that of the wild type(KD˜100 nM), placing the three residues with the greatest contributionto IGF-II binding all within the CD loop (see FIG. 2). The remaining CDloop mutation, Q1569A, caused only a minor reduction in affinity (KD=130nM). Of the mutations within the AB loop, only Y1542A had a greater than6 fold reduction in affinity (KD˜640 nM) compared to the wild type;S1543A and K1545A displayed a modest 2-3 fold affinity reduction (KD˜260nM and KD˜290 nM respectively). The S1596A mutation situated on the FGloop also resulted in only a minor reduction in affinity (KD˜170 nM).Intriguingly, the E1544A mutation on the AB loop resulted in an almost3-fold increase in IGF-II affinity (KD˜40 nM), providing indication thatthe acidic glutamate residue at this location is directly inhibitory tothe IGF-II interaction. The IGF-II binding kinetics of the G1619Rmutation, that mimics a common human polymorphism within domain 11 (60),were not significantly different to those of the wild type, providingindication that this polymorphism confers no functional consequence interms of IGF2 binding. This might be expected from its location on theGH loop, well away from the putative IGF2 binding site. In all cases,evaluation of sensograms revealed that significant changes in affinityappeared to be the result of concurrent changes in both association (ka)and dissociation (kd) rate constants.

Complete bias towards modification of either ‘on’ or the ‘off’ rates wasnot observed. During all of the above SPR experiments, analytes werealso passed over a separate flow cell on which biotinylated IGF1 hadbeen immobilised, to assess IGF2 specificity. In all cases binding wasof a very low level, insufficient to determine affinity.

SPR Analysis of Position 1544 Mutants of IGF2R Domain 11

Having established that replacement of glutamate at position 1544 withalanine caused an almost 3-fold increase in IGF-II affinity, via both‘on’ and ‘off’ rate modification, further mutations were generated atthis position in order to further evaluate this observation. The acidicglutamate residue was substituted for amino acid side chains of similarsize, with the hydrophobic residue valine (E1544V) and the basic residuelysine (E1544K). SPR analysis was then conducted as before, using thewild type domain 11 and the mutants E1544A, E1544V and E1544K asanalyte. Again, kinetic parameters and steady-state affinity data wereobtained from the resultant sensorgrams (FIG. 5 and Table 4).

Inspection of the sensorgrams shows that the activity of the immobilisedIGF-II sensor chip surface had reduced slightly with storage, with anapparent Rmax of ˜60 RU. Nevertheless, ample activity remained togenerate kinetic data. Like the E1544A mutant, the E1544V mutation alsoexhibited an, albeit slight, increase (˜2-fold) in affinity over thewild type. However, the E1544K mutant displayed a ˜6-fold increase inaffinity (KD˜15 nM) with a visibly decreased dissociation rate onsensograms (FIG. 5 d), and quantified by kinetic analysis (Table 4).This data confirms our novel observation that E1544 acts as anintra-domain negative regulator of the IGF-II interaction with IGF2Rdomain 11, and that conversion of the acidic glutamate residue to abasic residue (E1544K) has a significant additional enhancement ofaffinity above that of loss of the acidic side chain (E1544A). Thespecificity for IGF-II is retained in E1544 mutants, with the affinityfor IGF-I being still too low to be quantified. Moreover, it still showsthe same apparent rank order as the IGF2 affinity, as determined by themagnitude of the response at the highest analyte concentration, in thatE1544K gives the greatest response to IGF-I followed by the E1544A andE1544V mutants, with the wild type giving the least response.Site-directed mutagenesis was used to generate constructs in which E1544was replaced with all possible amino acid residues. SPR analysis wasconducted, using the purified domain 11 constructs, mutated at position1544, as analyte. Duplicate analyte injections were performed at aconcentration range of 4096-8 nM. Where possible, kinetic parameters andsteady-state affinity data were obtained from the resultant sensorgramsas before (Table 5 and FIG. 7). Protein yields obtained from the mutantin which cysteine had been introduced were very poor (˜1 mg/l) andsensorgrams from SPR analysis suggested a biphasic interaction thatcould not be easily interpreted. It seems likely that introduction of anadditional cysteine residue disrupted appropriate disulphide formation,resulting in reduced yields and misfolded protein.

The inclusion of a negatively charged acidic residue at position 1544 isdetrimental to affinity in this context, with both the wild type(glutamate) and aspartate conferring a relatively “poor” affinity(103.3±4.4 nM and 213.0±9.7 nM respectively). Interestingly, theaspartate residue results in a significant reduction in affinity whencompared to the wild type, predominantly due to an increase indissociation rate (14.00±0.10×10⁻² s⁻¹ over 7.87±0.29×10⁻² s⁻¹). In eachcase, replacement with the amide form (glutamine and asparagine) resultsin an improved affinity (26.7±2.3 nM and 118.0±5.6 nM respectively),although in the case of asparagine this only resulted in an affinityclose to that of the wild-type.

Of the remaining mutations all but three (P, G, N) resulted in anenhanced affinity when compared to the wild type (E). The remainingmutants displayed a relatively narrow affinity range (14.1±1.0 nM to60.0±3.0 nM). The reduction in affinity of both proline and glycinecontaining mutants probably reflects a structural perturbation aroundthe IGF-II binding site. Clearly, the greatest enhancement in affinity,above that of the wild type, was conferred by the residues lysine(16.9±0.2 nM, ˜6 fold affinity increase) and arginine (14.1±1.0 nM, ˜7fold affinity increase), providing indication that the interaction ofdomain 11 with IGF-II can be stabilised by replacement of the negativecharge at position 1544 with a positively charged residue, beyond thatwhich is achieved by removal of the negative charge. The affinityenhancing effect of the lysine residue was confirmed by ITC (26.4±5.1 nMfor E1544K). A considerable increase in affinity was also conferred byintroduction of a serine residue (19.7±1.5 nM ˜5 fold affinityincrease), predominantly through an association rate increase (k_(a) of21.20±0.21×10⁵ M⁻¹s⁻¹ compared to the wild type value of 6.62±0.13×10⁵M⁻¹s⁻¹).

Validation of BIAcore Affinity Data by Isothermal Titration Calorimetry

In order to validate the SPR sensorgram data, ITC was used to assess thebinding affinity between domain 11 and IGF-II. The affinity of theE1544A mutant was also assessed by ITC in order to confirm whether useof a second technique agreed as to the apparent increase in affinity(FIG. 6 and Table 6). Titrations were performed using 17×15 μlinjections of 40 μM IGF-II (after an initial 7.5 μl injection) into thereaction cell containing wild type domain 11 or the E1544A mutant. Aftersubtraction of the blank titration and fitting of the integrated bindingisotherms to a single site model, KD values were determined as being˜150 nM for wild-type domain 11 and ˜80 nM for E1544A. These values aresimilar to those obtained using SPR, 103 nM and 37 nM, respectively,indicating that both independent techniques closely represent the trueaffinities. The two fold differences may be simply accounted for by thefidelity of both techniques. Importantly, the ITC data agrees that theE1544A mutation confers a relative affinity increase of a magnitude thatis similar to that suggested by SPR (˜2 fold compared to ˜2.5 foldrespectively).

The affinity of the wild-type domain 11 approximated to 100-150 nM whenquantified using either SPR or ITC. The reproducibility and range of thesensogram data, combined with the use of the I1572T control, indicatedthat the described approach would have the sensitivity to accuratelyquantify subtle differences in ‘on’ and ‘off’ rates induced by sitedirected mutations. The validated affinity of IGF-II with domain 11alone is at least 10-100 fold less than full length IGF2R, with reportedaffinity constants ranging from ˜2 nM (13, 61) to 0.2 nM (62). Moreover,Domain 11 retains specificity for IGF-II compared to IGF-I, and theenhanced affinity of the intact receptor may be due to additionalslowing of the ‘off’ rate by domain 13, dimerisation of the full lengthreceptor and its affects on avidity, and additional as yet unidentifiedfactors (6, 15, 56, 63).

Domain 11-Fc Domain Fusions

Human IgG1 Fc domain C-terminally tagged fusion proteins of thewild-type, enhanced (11^(E1544K)) and null (11^(I1572A)) mutant forms ofIGF2R domain 11 (FIG. 8) were then generated. These constructs wereexpressed in Drosophila D.Mel-2 cells and secreted into the serum-freegrowth medium at a concentration of 10 mg per litre. A summary of thecalculated physical properties of the native Fc-tagged and untaggeddomain 11 proteins is shown in Table 7. A denaturing coomassie brilliantblue stained gel of the purified proteins showed that proteins ran as asingle band and that their denatured masses were very close to thosepredicted. Analytical gel filtration revealed that the native Fc-taggeddomain 11 proteins were dimeric and eluted as a single peak at 13.58 mlwith a mass (86 kDa), close to the predicted mass (88.06 kDa, see Table7).

Affinity of Fc-Tagged Domain 11 Proteins for IGF-II

The affinity of versions of Fc-tagged domain 11 fusion proteins, anddomain 11 constructs, for IGF-II were compared using surface plasmonresonance (BIAcore 3000). Attempts were made to immobilize Fc-taggeddomain 11 proteins upon a sensor chip surface (CM5 and CM5-proteinA) butwe encountered similar problems as described above with immobilisedmonomeric domain 11. Instead, immobilisation of biotinylated IGF-I andIGF-II on separate flow cells of a streptavidin coated sensor chip(BIAcore) generated reproducible sensorgram profiles when Fc-tagged anduntagged domain 11 proteins were passed over the surface as analytes.The Fc-tagged domain 11 kinetic data performed in triplicate generatedsensorgrams that were analyzed using a bivalent analyte model. Globalfits of the data with a bivalent analyte model without bulk refractiveindex change, generated no χ² greater than 1.94, and provided optimalfits to the data. As with the control monomeric domain 11 null mutant(11^(I1572A)), the 11^(1I1572A)-Fc homodimer had no affinity for eitherIGF-II or IGF-I. Moreover, neither Fc-tagged wild-type domain 11 (11-Fc)nor Fc-tagged enhanced mutant domain 11 (11^(E1544K)-Fc), had anaffinity for IGF-I, confirming that dimerisation had not altered ligandspecificity. From the sensorgrams for IGF-II binding, it was apparentthat 11-Fc and 11^(E1544K)-Fc both had high affinity for IGF-II, with Ruresponses for 11^(E1544K)-Fc at approximately twice the amplitude ofthose for 11-Fc at equal concentrations. Both Fc-tagged proteinsappeared to have reduced off-rates compared to the untagged monomers. Acomparison of these data shows that dimerisation by Fc-tagging increasedthe molar affinity (K_(D)) of wild-type domain 11 for IGF-II from118.8±3.5 nM to 3.26±0.3 nM, and the affinity of 11^(E1544K) from20.5±2.0 nM to 1.79±0.08 nM (Table 8). The 11^(E1544K)-Fc had thehighest affinity of all the proteins tested with the molar affinity of11^(E1544K)-Fc approximately twice that of 11-Fc. The improvement inaffinity of the Fc-tagged dimers compared to the monomers was largelydue to a substantial decrease in the off-rate (k_(d)1), from7.87±0.29×10⁻² s⁻¹ to 0.445±0.04×10⁻² s⁻¹ for 11-Fc, and from4.06±0.28×10⁻² s⁻¹ to 0.401±0.03×10⁻² s⁻¹ for 11^(E1544K)-Fc. Fc-taggingapproximately doubled the on-rate (k_(a)1) for wild-type domain 11 from6.62±0.13×10⁵ M⁻¹ s⁻¹ to 13.65±0.01×10⁵ M⁻¹ s⁻¹, but had little effecton the on-rate for 11^(E1544K), which remained high at 22.27±0.74×10⁵M⁻¹ s⁻¹ (Table 8). However, the substantially higher on-rate of11^(E1544K)-Fc compared to 11-Fc appeared to account for its highermolar affinity. The molar affinity of the Fc-tagged proteins wasconsidered too high to verify by isothermal titration calorimetry (ITC)as ITC cannot be easily used to measure affinities higher than 10 nM.Minor kinetic parameters were also obtained from the sensogram data(Table 8), but were too small to demonstrate any significant formationof the AB2 complex, where the analyte can form a bridge across twoligand molecules. This second binding event is purely a function ofligand immobilization. It is also worth noting that this is an exampleof a linked reaction, where the formation of the AB2 complex is entirelydependant upon the prior formation of AB, and that to dissociate, theAB2 must first decay back to AB. From the fits, the calculated R_(max)for the Fc-tagged proteins was approximately 75-85% of the theoreticalR_(max) (286 RU) and we concluded that a high percentage of theFc-tagged proteins were therefore functional. Increasing the valency ofdomain 11 was attempted following tetramerisation of biotinylatedversions of domain 11 using streptavidin. This resulted in protein thatcould not be easily purified, and it was not possible to quantify thefunctional affinity (avidity).

Ability of the Domain 11 Constructs to Block Functional Activity ofIGF-II In Vitro.

We next investigated the ability of the domain 11 proteins to inhibitthe actions of IGF-II in vitro, by assessing the phosphorylation ofdownstream targets of IGF1R signaling and IGF-II induced proliferationas measured by ³H-thymidine incorporation into nascent DNA. We used twodifferent cell lines, HaCaT human keratinocytes which have previouslybeen shown to proliferate in response to IGF-II (76, 77) andimmortalized Igf2^(−/−) mouse embryonic fibroblasts (Igf2^(−/−) MEFs)generated using a 3T3 protocol from an inbred 129S2 mouse line (78).Addition of IGF-II for 10 minutes to serum-starved HaCaT cells orIgf2^(−/−) MEFs led to an increase in the phosphorylation of PKB in adose dependent manner. Stimulation of cells with increasingconcentrations of IGF-II for 24 hours stimulated DNA synthesis, also ina dose dependent manner. Subsequent experiments were performed using 1.3nM IGF-II for signaling experiments and 6.5 nM IGF-II for proliferationexperiments, as these concentrations of IGF-II gave consistent maximalstimulation.

The domain 11 constructs 11^(wild-type), 11^(E1544K) (enhancedmutation), 11^(E1572A) (null mutation) 11-Fc, 11^(E1544K)-Fc and11^(E1572A)-Fc were investigated for their ability to blockIGF-II-stimulated proliferation and signaling in Igf2^(−/−)MEFs. Of thedifferent constructs, only the Fc tagged enhanced mutant(11^(E1544K)-Fc) showed significant ability to inhibit IGF-II-stimulatedproliferation and activation of the IGF1R (FIGS. 11 and 12). Whenequivalent numbers of IGF-II binding sites were present for the single11^(E1544K) domain, compared to the Fc tagged homodimer, only11^(E1544K)-Fc could inhibit IGF-II mediated activation of the cellssignificantly, even though it appears that both proliferation andsignaling are slightly attenuated by 11^(E1544K) (FIG. 11).11^(E1544K)-Fc was also the construct with the highest measured affinityfor IGF-II (Table 8).

The inhibitory properties of 11^(E1544K)-Fc were further investigated byassessing its ability to inhibit IGF-II dependent proliferation in HaCaTcells (FIG. 13). Keeping the concentration of IGF-II constant (6.5 nM)we found that 11^(E1544K)-Fc decreased the ability of IGF-II tostimulate proliferation in a dose dependent manner, with 650 nM and 1300nM significantly decreasing proliferation by 50% (p=0.005) and 73%(p=0.013) respectively (FIG. 13). When the decrease in 3H-thymidineuptake was equated to the concentration of functional IGF-II remainingin the media, 650 nM and 1300 nM 11^(E1544K)-Fc reduced the amount ofactive IGF-II by 82% and 90% respectively (see inset FIG. 13).

Compared to 11^(E1544K)-Fc, the Fc tagged null mutant (11^(I1572A)-Fc)that had no affinity for IGF-II, was unable to inhibit either IGF-IIstimulated proliferation (FIG. 14) or stimulation of IGF1R activation asmeasured by phosphorylation of PKB (Akt). This provides indication thatthe ability of 11^(E1544K)-Fc to inhibit the actions of IGF-II isdependent on its capacity to bind directly to the ligand. In addition,11^(E1544K)-Fc inhibitory function was specific for IGF-II, as it wasunable to block the actions of IGF-I either on proliferation orstimulation via IGF1R.

Finally, the inhibition of chemotherapy (doxorubicin) induced apoptosisand caspase activation by IGF-II was also attenuated by11^(I1572A)-Fc^(a). Overall, the surface plasmon resonance resultscombined with cell function assays, indicate that 11^(E1544K)-Fc is bothhighly specific for IGF-II and that its inhibitory potency reflects itsenhanced affinity for the IGF-II ligand.

These data present the first systematic mutational analysis of the IGF2Rdomain 11 binding site for IGF-II, combined with detailed and validatedSPR analysis of real time binding kinetics. The previously identified CDloop mutation (I1572T), and the new mutation identified here (F1567A)appear to form the hydrophobic core of the ligand binding site. Bothmutations abolish binding, and are the only two hydrophobic residueswithin the AB and CD loops.

For mutations of non-hydrophobic residues, SPR technology was able toquantify the relative impact of each side chain to the overall affinity.In this context, the predominant changes were detected by IGF2R domain11 mutagenesis and IGF-II binding kinetics in the FG and AB loops, wherein the latter adjacent residues were able to either enhance or reduceaffinity (e.g. S1543 and E1544). Rather than a specific alteration ineither the ‘on’ (association) or the ‘off’ (dissociation) rate, theinteraction of IGF-II with mutated versions of domain 11 appeared tolead to relatively equal effects on both the ‘on’ and the ‘off’ rates.Most of these residues are referred to as ‘second sphere’, ‘latch’ or‘O’ ring residues that can substantially modify affinity in an additivefashion, and so stabilise the interaction via hydrogen bonds and saltbridges. Here, we identify a novel function for E1544 as a second sphereresidue that acts as a negative regulator, with loss of the acidic sidechain leading to enhanced affinity (E1544A). Further enhancement ofaffinity over that seen for E1544A, was established by conversion of theglutamate residue to a similar sized basic residue (E1544K).Importantly, the other residues of the AB loop failed to contribute anegative regulatory role. The AB loop residue E1544 is also conserved inmammalian species, and absent in chicken (64). As the normal function ofIGF2R is to clear IGF-II via binding and internalisation, theidentification of an amino acid residue that has evolved to negativelyregulate this affinity is an unexpected finding.

The tumour suppressor function of IGF2R depends on its bindinginteraction with IGF-II, as expression of a soluble IGF2R full lengthreceptor rescues the growth promotion consequences of biallelicexpression of Igf2 in the ApcMin/+ mouse model of intestinal adenoma.Thus, the supply of soluble high affinity forms of IGF2R may be usefulto target and treat the consequences of increased IGF-II in cancer. Themolecules described herein may be useful in the development of highaffinity therapeutic traps for IGF-II.

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TABLE 1 Oligo- nucleotide Name Sequence (5′ to 3′) Y1542AFCGGGATTCACAGCTGCTgcCAGCGAGAAGGGGTTGG (SEQ ID NO: 16) Y1542ARCCAACCCCTTCTCGCTGgcAGCAGCTGTGAATCCCG (SEQ ID NO: 17) S1543AFCGGGATTCACAGCTGCITACgcCGAGAAGGGGTTGG (SEQ ID NO: 18) S1543ARCCAACCCCTTCTCGgcGTAAGCAGCTGTGAATCCCG (SEQ ID NO: 19) E1544AFCACAGCTGCTTACAGCGcGAAGGGGTTGGTTTAC (SEQ ID NO: 20) E1544ARGTAAACCAACCCCTTCgCGCTGTAAGCAGCTGTG (SEQ ID NO: 21) K1545AFCAGCTGCTTACAGCGAGgcGGGGTTGGTTTACATGAGC (SEQ ID NO: 22) K1545ARGCTCATGTAAACCAAGCCCgcCTCGCTGTAAGCAGCTG (SEQ ID NO: 23) F1567AFCCTGGCGTGGGGGCCTGCgcTGGACAGACCAGGATTAGC (SEQ ID NO: 24) F1567ARGCTAATCCTGGTCTGTCCAgcGCAGGCCCCCACGCCAGG (SEQ ID NO: 25) Q1569AFCGTGGGGGCCTGCTTTGGAgcGACCAGGATTAGCGTGG (SEQ ID NO: 26) Q1569ARCCACGCTAATCCTGGTCgcTCCAAAGCAGGCCCCCACG (SEQ ID NO: 27) T1570AFGCCTGCTTTGGACAGgCCAGGATTAGCGTGGGC (SEQ ID NO: 28) T1570ARGCCCACGCTAATCCTGGcCTGTCCAAAGCAGGC (SEQ ID NO: 29) I1572AFGCTTTGGACAGACCAGGgcTAGCGTGGGCAAGGCC (SEQ ID NO: 30) I1572ARGGCCTTGCCCACGCTAgcCCTGGTCTGTCCAAAGC (SEQ ID NO: 31) I1572TFGCTTTGGACAGACCAGGAcTAGCGTGGGCAAGGCC (SEQ ID NO: 32) I1572TRGGCCTTGCCCACGCTAgTCCTGGTCTGTCGAAAGC (SEQ ID NO: 33) S1596AEGGTGTACAAGGATGGGgCCCCTTGTCCCTCCAAATCC (SEQ ID NO: 34) S1596ARGGATTTGGAGGGACAAGGGGcCCCATCCTTGTACACC (SEQ ID NO: 35) G1619RFCGTGTGCAGGCCTGAGGCCaGGCCAACCAATAGGCC (SEQ ID NO: 36) G1619RRGGCCTATTGGTTGGCCtGGCCTCAGGCCTGCACACG (SEQ ID NO: 37) E1544VFCACAGCTGCTTACAGCGtGAAGGGGTTGGTTTAC (SEQ ID NO: 38) E1544VRGTAAACCAACCCCTTCaCGCTGTAAGCAGCTGTG (SEQ ID NO: 39) E1544KFCACAGCTGCTTACAGCaAGAAGGGGTTGGTTTAC (SEQ ID NO: 40) E1544KRGTAAACCAACCCCTTCTtGCTGTAAGCAGCTGTG (SEQ ID NO: 41)

TABLE 2 Source of Kinetic Parameters Steady state Domain 11 k_(a) (×10⁵M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (×10⁻⁹ M) K_(D) (×10⁻⁹ M) E. coli 5.53 ± 0.250.063 ± 0.003 114.0 ± 5.0 114 ± 5.4 Pichia pastoris 5.25 ± 0.35 0.053 ±0.002 101.8 ± 3.6 103 ± 4.4

TABLE 3 Kinetic Parameters Steady state Relative Loop Mutation k_(a)(×10⁵ M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (×10⁻⁹ M) K_(D) (×10⁻⁹ M) Affinity WT5.25 ± 0.35 0.053 ± 0.002 102 ± 4 103 ± 4 1.00 AB Y1542A 2.65 ± 0.130.169 ± 0.008 639 ± 5 633 ± 6 0.16 S1543A 3.69 ± 0.16 0.096 ± 0.003 260± 4 262 ± 3 0.39 E1544A 9.34 ± 1.04 0.033 ± 0.002  36 ± 2  37 ± 3 2.79K1545A 2.73 ± 0.15 0.078 ± 0.004 287 ± 8  289 ± 10 0.36 CD F1567A — — —— — Q1569A 5.29 ± 0.19 0.069 ± 0.001 130 ± 4 130 ± 3 0.80 T1570A 2.95 ±0.03 0.281 ± 0.002 954 ± 8  930 ± 12 0.11 I1572A — — — — — I1572T — — —— — FG S1596A 4.41 ± 0.15 0.079 ± 0.004 179 ± 2 170 ± 4 0.61 GH G1619R4.39 ± 0.42 0.050 ± 0.003 114 ± 5 114 ± 4 0.90

TABLE 4 Kinetic Parameters Steady state Relative Mutation k_(a) (×10⁵M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (×10⁻⁹ M) K_(D) (×10⁻⁹ M) Affinity WT 5.25 ±0.35 0.053 ± 0.002 101.8 ± 3.6  103.3 ± 4.4  1.00 E1544A 9.34 ± 1.040.033 ± 0.002 35.9 ± 2.3 37.0 ± 2.6 2.79 E1544V 8.37 ± 0.42 0.041 ±0.001 49.8 ± 2.7 52.7 ± 3.1 1.96 E1544K 15.20 ± 0.15  0.023 ± 0.000 15.2± 0.1 16.9 ± 0.2 6.12

TABLE 5 Residue at Side-chain position Kinetic Parameters Steady stateRelative property 1544 k_(a) (×10⁵ M⁻¹s⁻¹) k_(d) (×10⁻² s⁻¹) K_(D)(×10⁻⁹ M) K_(D) (×10⁻⁹ M) Affinity Hydrophobic A 12.30 ± 0.52 5.58 ±0.18 45.5 37.0 ± 2.6 2.79 V 11.23 ± 0.67 6.75 ± 0.15 60.5 52.7 ± 3.11.96 L 10.50 ± 0.04 6.55 ± 0.05 62.4 51.1 ± 1.7 2.02 I 13.00 ± 0.05 6.49± 0.06 49.9 42.5 ± 2.8 2.43 P  7.34 ± 0.03 17.20 ± 0.08  234.3 211.0 ±8.9  0.49 W  9.36 ± 0.05 5.88 ± 0.05 62.8 53.2 ± 2.2 1.94 F 11.40 ± 0.087.91 ± 0.11 69.4 56.4 ± 2.6 1.83 M 12.70 ± 0.08 5.66 ± 0.06 44.6 37.6 ±1.9 2.75 Polar G 11.00 ± 0.09 21.90 ± 0.22  199.1 180.0 ± 7.3  0.57 S21.20 ± 0.21 4.94 ± 0.10 23.3 19.7 ± 1.5 5.24 T  9.98 ± 0.07 6.92 ± 0.0769.3 60.0 ± 3.0 1.72 Y  9.44 ± 0.03 6.14 ± 0.01 65.0 57.4 ± 2.5 1.80 C —— — — — N  8.67 ± 0.07 12.20 ± 0.15  140.7 118.0 ± 5.6  0.88 Q 17.60 ±0.19 5.78 ± 0.12 32.8 26.7 ± 2.3 3.87 Basic K 20.23 ± 2.97 4.06 ± 0.2820.5 16.9 ± 0.2 6.12 R 22.90 ± 0.06 4.07 ± 0.04 17.8 14.1 ± 1.0 7.33 H18.00 ± 0.16 6.89 ± 0.10 38.3 33.7 ± 2.3 3.07 Acidic D  6.08 ± 0.0314.00 ± 0.10  230.3 213.0 ± 9.7  0.48 E (WT)  6.62 ± 0.13 7.87 ± 0.29118.8 103.3 ± 4.4  1.00

TABLE 6 Domain 11 Affinity, K_(D) (×10⁻⁹ M) construct Biacore ITC WT103.3 ± 4.4 149.6 ± 4.4  E1544A  37.0 ± 2.6 81.4 ± 9.0 E1544K  16.9 ±0.2 26.4 ± 5.1 Q1569A 129.7 ± 2.7 208.9 ± 13.0 S1596A 170.0 ± 3.6 323.6± 42.0

TABLE 7 Molecular Weight Ext. coefficient Protein (kDa) PI (M⁻¹ cm⁻¹@280mm) 11^(Wild type) 16.82 7.76 13650 11^(E1544K) ″ 8.45 ″ 11^(I1572A)16.77 7.76 ″ 11^(Wild type) -Fc 88.06 8.02 94420 11^(E1544K) ″ 8.36 ″11^(I1572A) 87.98 8.02 ″

TABLE 8 Major Kinetic Parameters Minor Kinetic Parameters ka1 kd1 K_(D)k_(a)2 k_(a)2 k_(d)2 Protein (×10⁵M⁻¹s⁻¹) (×10⁻² s⁻¹) (×10⁻⁹M) (×10⁻³s⁻¹) (×10⁻⁵RU⁻¹s⁻¹) (×10⁻⁴ s⁻¹) 11^(Wild type)  6.62 ± 0.13  7.87 ± 0.29118.8 ± 3.5  2.45 ± 0.33 — 121 ± 7.2  11^(E1544K) 20.23 ± 2.97  4.06 ±0.28 20.5 ± 2.0 3.83 ± 1.10 — 79 ± 28 11^(I1572A) — — — — — —11^(Wild type)-Fc 13.65 ± 0.01 0.445 ± 0.04 3.26 ± 0.3 — 7.12 ± 0.192.33 ± 0.12 11^(E1544K) 22.27 ± 0.74 0.401 ± 0.03  1.79 ± 0.08 — 7.86 ±0.04 1.26 ± 0.03 11^(I1572A) — — — — — —

Sequences SEQ ID NO: 1 1mgaaagrsph lgpaparrpq rsllllqlll lvaapgstqa qaapfpelcs ytweavdtkn 61nvlykinicg svdivqcgps savcmhdlkt rtyhsvgdsv lrsatrslle fnttvscdqq 121gtnhrvqssi aflcgktlgt pefvtatecv hyfewrttaa ckkdifkank evpcyvfdee 181lrkhdlnpli klsgaylvdd sdpdtslfin vcrdidtlrd pgsqlracpp gtaaclvrgh 241qafdvgqprd glklvrkdrl vlsyvreeag kldfcdghsp avtitfvcps erregtipkl 301taksncryei ewiteyachr dylesktcsl sgeqqdvsid ltplaqsggs syisdgkeyl 361fylnvcgete iqfcnkkqaa vcqvkksdts qvkaagryhn qtlrysdgdl tliyfggdec 421ssgfqrmsvi nfecnktagn dgkgtpvftg evdctyfftw dteyacvkek edllcgatdg 481kkrydlsalv rhaepeqnwe avdgsqtete kkhffinich rvlqegkarg cpedaavcav 541dkngsknlgk fisspmkekg niqlsysdgd dcghgkkikt nitlvckpgd lesapvlrts 601geggcfyefe wrtaaacvls ktegenctvf dsqagfsfdl spltkkngay kvetkkydfy 661invcgpvsvs pcqpdsgacq vaksdektwn lglsnaklsy ydgmiqlnyr ggtpynnerh 721tpratlitfl cdrdagvgfp eyqeednsty nfrwytsyac peeplecvvt dpstleqydl 781sslakseggl ggnwyamdns gehvtwrkyy invcrplnpv pgcnryasac qmkyekdqgs 841ftevvsisnl gmaktgpvve dsgsllleyv ngsacttsdg rqttyttrih lvcsrgrlns 901hpifslnwec vvsflwntea acpiqtttdt dqacsirdpn sgfvfnlnpl nssqgynvsg 961igkifmfnvc gtmpvcgtil gkpasgceae tqteelknwk parpvgieks lqlstegfit 1021ltykgplsak gtadafivrf vcnddvysgp lkflhqdids gqgirntyfe fetalacvps 1081pvdcqvtdla gneydltgls tvrkpwtavd tsvdgrkrtf ylsvcnplpy ipgcqgsavg 1141sclvsegnsw nlgvvqmspq aaangslsim yvngdkcgnq rfstritfec aqisgspafq 1201lqdgceyvfi wrtveacpvv rvegdncevk dprhgnlydl kplglndtiv sageytyyfr 1261vcgklssdvc ptsdkskvvs scqekrepqg fhkvaglltq kltyengllk mnftggdtch 1321kvyqrstaif fycdrgtqrp vflketsdcs ylfewrtqya cppfdltecs fkdgagnsfd 1381lsslsrysdn weaitgtgdp ehylinvcks lapqagtepc ppeaaacllg gskpvnlgrv 1441rdgpqwrdgi ivlkyvdgdl cpdgirkkst tirftcsesq vnsrpmfisa vedceytfaw 1501ptatacpmks nehddcqvtn pstghlfdls slsgragfta aysekglvym sicgenencp 1561pgvgacfgqt risvgkankr lryvdqvlql vykdgspcps ksglsyksvi sfvcrpeagp 1621tnrpmlisld kqtctlffsw htplaceqat ecsvrngssi vdlsplihrt ggyeaydese 1681ddasdtnpdf yinicqplnp mhavpcpaga avckvpidgp pidigrvagp plinpianei 1741ylnfesstpc ladkhfnyts liafhckrgv smgtpkllrt secdfvfewe tpvvcpdevr 1801mdgctltdeq llystnlssl ststfkvtrd srtysvgvct favgpeqggc kdggvcllsg 1861tkgasfgrlq smkldyrhqd eavvlsyvng drcppetddg vpcvfpfifn gksyeeciie 1921sraklwcstt adydrdhewg fcrhsnsyrt ssiifkcded edigrpqvfs evrgcdvtfe 1981wktkvvcppk kleckfvqkh ktydlrllss ltgswslvhn gvsyyinlcq kiykgplgcs 2041erasicrrtt tgdvqvlglv htqklgvigd kvvvtyskgy pcggnktass vieltctktv 2101grpafkrfdi dsctyyfswd sraacavkpq evqmvngtit npingksfsl gdiyfklfra 2161sgdmrtngdn ylyeiqlssi tssrnpacsg anicqvkpnd qhfsrkvgts dktkyylqdg 2221dldvvfasss kcgkdktksv sstiffhcdp lvedgipefs hetadcqylf swytsavcpl 2281gvgfdsenpg ddgqmhkgls ersqavgavl slllvaltcc llalllykke rretvisklt 2341tccrrssnvs ykyskvnkee etdenetewl meeiqlpppr qgkegqengh ittksvkals 2401slhgddqdse devltipevk vhsgrgagae sshpvrnaqs nalqereddr vglvrgekar 2461kgksssaqqk tvsstklvsf hddsdedllh i SEQ ID NO: 2 1cgagcccagt cgagccgcgc tcacctcggg ctcccgctcc gtctccacct ccgcctttgc 61cctggcggcg cgaccccgtc ccggcgcggc ccccagcagt cgcgcgccgt tagcctcgcg 121cccgccgcgc agtccgggca cggcgcgatg ggggccgccg ccggccggag cccccacctg 181gggcccgcgc ccgcccgccg cccgcagcgc tctctgctcc tgctgcagct gctgctgctc 241gtcgctgccc cggggtccac gcaggcccag gccgccccgt tccccgagct gtgcagttat 301acatgggaag ctgttgatac caaaaataat gtactttata aaatcaacat ctgtggaagt 361gtggatattg tcoagtgcgg gccatcaagt gctgtttgta tgcacgactt gaagacacgc 421acttatcatt cagtgggtga ctatgttttg agaagtgcaa ccagatctct cctggaattc 481aacacaacag tgagctgtga ccagcaaggc acaaatcaca gagtccagag cagcattgcc 541ttcctgtgtg ggaaaaccct gggaactcct gaatttgtaa ctgcaacaga atgtgtgcac 601tactttgagt ggaggaccac tgcagcctgc aagaaagaca tatttaaagc aaataaggag 661gtgccatgct atgtgtttga tgaagagttg aggaagcatg atctcaatcc tctgatcaag 721cttagtggtg cctacttggt ggatgactcc gatccggaca cttctctatt cataaatgtt 781tgtagagaca tagacacact acgagaccca ggttcacagc tgcgggcctg tccccccggc 841actgccgcct gcctggtaag aggacaccag gcgtttgatg ttggccagcc ccgggacgga 901ctgaagctgg tgcgcaagga caggcttgtc ctgagttacg tgagggaaga ggcaggaaag 961ctagactttt gtgatggtca cagccctgcg gtgactatta catttgtttg cccgtcggag 1021cggagagagg gcaccattcc caaactcaca gctaaatcca actgccgcta tgaaattgag 1081tggattactg agtatgcctg ccacagagat tacctggaaa gtaaaacttg ttctctgagc 1141ggcgagcagc aggatgtctc catagacctc acaccacttg cccagagcgg aggttcatcc 1201tatatttcag atggaaaaga atatttgttt tatttgaatg tctgtggaga aactgaaata 1261cagttctgta ataaaaaaca agctgcagtt tgccaagtga aaaagagcga tacctctcaa 1321gtcaaagcag caggaagata ccacaatcag accctccgat attcggatgg agacctcacc 1381ttgatatatt ttggaggtga tgaatgcagc tcagggtttc agcggatgag cgtcataaac 1441tttgagtgca ataaaaccgc aggtaacgat gggaaaggaa ctcctgtatt cacaggggag 1501gttgactgca cctacttctt cacatgggac acggaatacg cctgtgttaa ggagaaggaa 1561gacctcctct gcggtgccac cgacgggaag aagcgctatg acctgtccgc gctggtccgc 1621catgcagaac cagagcagaa ttgggaagct gtggatggca gtcagacgga aacagagaag 1681aagcattttt tcattaatat ttgtcacaga gtgctgcagg aaggcaaggc acgagggtgt 1741cccgaggacg cggcagtgtg tgcagtggat aaaaatggaa gtaaaaatct gggaaaattt 1801atttcctctc ccatgaaaga gaaaggaaac attcaactct cttattcaga tggtgatgat 1861tgtggtcatg gcaagaaaat taaaactaat atcacacttg tatgcaagcc aggtgatctg 1921gaaagtgcac cagtgttgag aacttctggg gaaggcggtt gcttttatga gtttgagtgg 1981cgcacagctg cggcctgtgt gctgtctaag acagaagggg agaactgcac ggtctttgac 2041tcccaggcag ggttttcttt tgacttatca cctctcacaa agaaaaatgg tgcctataaa 2101gttgagacaa agaagtatga cttttatata aatgtgtgtg gcccggtgtc tgtgagcccc 2161tgtcagccag actcaggagc ctgccaggtg gcaaaaagtg atgagaagac ttggaacttg 2221ggtctgagta atgcgaagct ttcatattat gatgggatga tccaactgaa ctacagaggc 2281ggcacaccct ataacaatga aagacacaca ccgagagcta cgctcatcac ctttctctgt 2341gatcgagacg cgggagtggg cttccctgaa tatcaggaag aggataactc cacctacaac 2401ttccggtggt acaccagcta tgcctgcccg gaggagcccc tggaatgcgt agtgaccgac 2461ccctccacgc tggagcagta cgacctctcc agtctggcaa aatctgaagg tggccttgga 2521ggaaactggt atgccatgga caactcaggg gaacatgtca cgtggaggaa atactacatt 2581aacgtgtgtc ggcctctgaa tccagtgccg ggctgcaacc gatatgcatc ggcttgccag 2641atgaagtatg aaaaagatca gggctccttc actgaagtgg tttccatcag taacttggga 2701atggcaaaga ccggcccggt ggttgaggac agcggcagcc tccttctgga atacgtgaat 2761gggtcggcct gcaccaccag cgatggcaga cagaccacat ataccacgag gatccatctc 2821gtctgctcca ggggcaggct gaacagccac cccatctttt ctctcaactg ggagtgtgtg 2881gtcagtttcc tgtggaacac agaggctgcc tgtcccattc agacaacgac ggatacagac 2941caggcttgct ctataaggga tcccaacagt ggatttgtgt ttaatcttaa tccgctaaac 3001agttcgcaag gatataacgt ctctggcatt gggaagattt ttatgtttaa tgtctgcggc 3061acaatgcctg tctgtgggac catcctggga aaacctgctt ctggctgtga ggcagaaacc 3121caaactgaag agctcaagaa ttggaagcca gcaaggccag tcggaattga gaaaagcctc 3181cagctgtcca cagagggctt catcactctg acctacaaag ggcctctctc tgccaaaggt 3241accgctgatg cttttatcgt ccgctttgtt tgcaatgatg atgtttactc agggcccctc 3301aaattcctgc atcaagatat cgactctggg caagggatcc gaaacactta ctttgagttt 3361gaaaccgcgt tggcctgtgt tccttctcca gtggactgcc aagtcaccga cctggctgga 3421aatgagtacg acctgactgg cctaagcaca gtcaggaaac cttggacggc tgttgacacc 3481tctgtcgatg ggagaaagag gactttctat ttgagcgttt gcaatcctct cccttacatt 3541cctggatgcc agggcagcgc agtggggtct tgcttagtgt cagaaggcaa tagctggaat 3601ctgggtgtgg tgcagatgag tccccaagcc gcggcgaatg gatctttgag catcatgtat 3661gtcaacggtg acaagtgtgg gaaccagcgc ttctccacca ggatcacgtt tgagtgtgct 3721cagatatcgg gctcaccagc atttcagctt caggatggtt gtgagtacgt gtttatctgg 3781agaactgtgg aagcctgtcc cgttgtcaga gtggaagggg acaactgtga ggtgaaagac 3841ccaaggcatg gcaacttgta tgacctgaag cccctgggcc tcaacgacac catcgtgagc 3901gctggcgaat acacttatta cttccgggtc tgtgggaagc tttcctcaga cgtctgcccc 3961acaagtgaca agtccaaggt ggtctcctca tgtcaggaaa agcgggaacc gcagggattt 4021cacaaagtgg caggtctcct gactcagaag ctaacttatg aaaatggctt gttaaaaatg 4081aacttcacgg ggggggacac ttgccataag gtttatcagc gctccacagc catcttcttc 4141tactgtgacc gcggcaccca gcggccagta tttctaaagg agacttcaga ttgttcctac 4201ttgtttgagt ggcgaacgca gtatgcctgc ccacctttcg atctgactga atgttcattc 4261aaagatgggg ctggcaactc cttcgacctc tcgtccctgt caaggtacag tgacaaatgg 4321gaagccatca ctgggacggg ggacccggag cactacctca tcaatgtctg caagtctctg 4381gccccgcagg ctggcactga gccgtgccct ccagaagcag ccgcgtgtct gctgggtggc 4441tccaagcccg tgaacctcgg cagggtaagg gacggacctc agtggagaga tggcataatt 4501gtcctgaaat acgttgatgg cgacttatgt ccagatggga ttcggaaaaa gtcaaccacc 4561atccgattca cctgcagcga gagccaagtg aactccaggc ccatgttcat cagcgccgtg 4621gaggactgtg agtacacctt tgcatggccc acagccacag cctgtcccat gaagagcaac 4681gagcatgatg actgccaggt caccaaccca agcacaggac acotgtttga tctgagctcc 4741ttaagtggca gggcgggatt cacagctgct tacagcgaga aggggttggt ttacatgagc 4801atctgtgggg agaatgaaaa ctgccctcct ggcgtggggg cctgctttgg acagaccagg 4861attagcgtgg gcaaggccaa caagaggctg agatacgtgg accaggtcct gcagctggtg 4921tacaaggatg ggtccccttg tccctccaaa tccggcctga gctataagag tgtgatcagt 4981ttcgtgtgca ggcctgaggc cgggccaacc aataggccca tgctcatctc cctggacaag 5041cagacatgca ctctcttctt ctcctggcac acgccgctgg cctgcgagca agcgaccgaa 5101tgttccgtga ggaatggaag ctctattgtt gacttgtctc cccttattca tcgcactggt 5161ggttatgagg cttatgatga gagtgaggat gatgcctccg ataccaaccc tgatttctac 5221atcaatattt gtcagccact aaatcccatg cacgcagtgc cctgtcctgc cggagccgct 5281gtgtgcaaag ttcctattga tggtcccccc atagatatcg gccgggtagc aggaccacca 5341atactcaatc caatagcaaa tgagatttac ttgaattttg aaagcagtac tccttgctta 5401gcggacaagc atttcaacta cacctcgctc atcgcgtttc actgtaagag aggtgtgagc 5461atgggaacgc ctaagctgtt aaggaccagc gagtgcgact ttgtgttcga atgggagact 5521cctgtcgtct gtcctgatga agtgaggatg gatggctgta ccctgacaga tgagcagctc 5581ctctacagct tcaacttgtc cagcctttcc acgagcacct ttaaggtgac tcgcgactcg 5641cgcacctaca gcgttggggt gtgcaccttt gcagtcgggc cagaacaagg aggctgtaag 5701gacggaggag tctgtctgct ctcaggcacc aagggggcat cctttggacg gctgcaatca 5761atgaaactgg attacaggca ccaggatgaa gcggtcgttt taagttacgt gaatggtgat 5821cgttgccctc cagaaaccga tgacggcgtc ccctgtgtct tccccttcat attcaatggg 5881aagagctacg aggagtgcat catagagagc agggcgaagc tgtggtgtag cacaactgcg 5941gactacgaca gagaccacga gtggggcttc tgcagacact caaacagcta ccggacatcc 6001agcatcatat ttaagtgtga tgaagatgag gacattggga ggccacaagt cttcagtgaa 6061gtgcgtgggt gtgatgtgac atttgagtgg aaaacaaaag ttgtctgccc tccaaagaag 6121ttggagtgca aattcgtcca gaaacacaaa acctacgacc tgcggctgct ctcctctctc 6181accgggtcct ggtccctggt ccacaacgga gtctcgtact atataaatct gtgccagaaa 6241atatataaag ggcccctggg ctgctctgaa agggccagca tttgcagaag gaccacaact 6301ggtgacgtcc aggtcctggg actcgttcac acgcagaagc tgggtgtcat aggtgacaaa 6361gttgttgtca cgtactccaa aggttatccg tgtggtggaa ataagaccgc atcctccgtg 6421atagaattga cctgtacaaa gacggtgggc agacctgcat tcaagaggtt tgatatcgac 6481agctgcactt actacttcag ctgggactcc cgggctgcct gcgccgtgaa gcctcaggag 6541gtgcagatgg tgaatgggac catcaccaac cctataaatg gcaagagctt cagcctcgga 6601gatatttatt ttaagctgtt cagagcctct ggggacatga ggaccaatgg ggacaactac 6661ctgtatgaga tccaactttc ctccatcaca agctccagaa acccggcgtg ctctggagcc 6721aacatatgcc aggtgaagcc caacgatcag cacttcagtc ggaaagttgg aacctctgac 6781aagaccaagt actaccttca agacggcgat ctcgatgtcg tgtttgcctc ttcctctaag 6841tgcggaaagg ataagaccaa gtctgtttct tccaccatct tcttccactg tgaccctctg 6901gtggaggacg ggatccccga gttcagtcac gagactgccg actgccagta cctcttctct 6961tggtacacct cagccgtgtg tcctctgggg gtgggctttg acagcgagaa tcccggggac 7021gacgggcaga tgcacaaggg gctgtcagaa cggagccagg cagtcggcgc ggtgctcagc 7081ctgctgctgg tggcgctcac ctgctgcctg ctggccctgt tgctctacaa gaaggagagg 7141agggaaacag tgataagtaa gctgaccact tgctgtagga gaagttccaa cgtgtcctac 7201aaatactcaa aggtgaataa ggaagaagag acagatgaga atgaaacaga gtggctgatg 7261gaagagatcc agctgcctcc tccacggcag ggaaaggaag ggcaggagaa cggccatatt 7321accaccaagt cagtgaaagc cctcagctcc ctgcatgggg atgaccagga cagtgaggat 7381gaggttctga ccatcccaga ggtgaaagtt cactcgggca ggggagctgg ggcagagagc 7441tcccacccag tgagaaacgc acagagcaat gcccttcagg agcgtgagga cgatagggtg 7501gggctggtca ggggtgagaa ggcgaggaaa gggaagtcca gctctgcaca gcagaagaca 7561gtgagctcca ccaagctggt gtccttccat gacgacagcg acgaggacct cttacacatc 7621tgactccgca gtgcctgcag gggagcacgg agccgcggga cagccaagca cctccaaaca 7681aataagactt ccactcgatg atgcttctat aattttgcct ttaacagaaa ctttcaaaag 7741ggaagagttt ttgtgatggg ggagagggtg aaggaggtca ggccccactc cttcctgatt 7801gtttacagtc attggaataa ggcatggctc agatcggcca cagggcggta ccttgtgccc 7861agggttttgc cccaagtcct catttaaaag cataaggccg gacgcatctc aaaacagagg 7921gctgcattcg aagaaaccct tgctgcttta gtcccgatag ggtatttgac cccgatatat 7981tttagcattt taattctctc cccctattta ttgactttga caattactca ggtttgagaa 8041aaaggaaaaa aaaacagcca ccgtttcttc ctgccagcag gggtgtgatg taccagtttg 8101tccatcttga gatggtgagg ctgtcagtgt atggggcagc ttccggcggg atgttgaact 8161ggtcattaat gtgtcccctg agttggagct cattctgtct cttttctctt ttgctttctg 8221tttcttaagg gcacacacac gtgcgtgcga gcacacacac acatacgtgc acagggtccc 8281cgagtgccta ggttttggag agtttgcctg ttctatgcct ttagtcagga atggctgcac 8341ctttttgcat gatatcttca agcctgggcg tacagagcac atttgtcagt atttttgccg 8401gctggtgaat tcaaacaacc tgcccaaaga ttgatttgtg tgtttgtgtg tgtgtgtgtg 8461tgtgtgtgtg tgtgtgagtg gagttgaggt gtcagagaaa atgaattttt tccagatttg 8521gggtataggt ctcatctctt caggttctca tgataccacc tttactgtgc ttattttttt 8581aagaaaaaag tgttgatcaa ccattcgacc tataagaagc cttaatttgc acagtgtgtg 8641acttacagaa actgcatgaa aaatcatggg ccagagcctc ggccctagca ttgcacttgg 8701cctcatgctg gagggaggct gggcgggtac agcgcggagg aggagggagg ccaggcgggc 8761atggcgtgga ggaggaggga ggccgggcgg tcacagcatg gaggaggagg gaggcgctgc 8821tggtgttctt aitctggcgg cagcgccttt cctgccatgt ttagtgaatg acttttctcg 8881cattgtagaa ttgtatatag actctggtgt tctattgctg agaagcaaac cgccctgcag 8941catccctcag cctgtaccgg tttggctggc ttgtttgatt tcaacatgag tgtatttttt 9001aaaattgatt tttctcttca tttttttttc aatcaacttt actgtaatat aaagtattca 9061acaatttcaa taaaagataa attattaaaa SEQ ID NO: 3 1nehddcqvtn pstghlfdls slsgragfta aysekglvym sicgenencp pgvgacfgqt 61risvgkankr lryvdqvlql vykdgspcps ksglsyksvi sfvcrpeagp tnrpmlisld 121kqtctlffsw htplaceqat

1. A mutant IGF-II binding domain comprising an amino acid sequencewhich has at least 95% sequence identity with residues 1511 to 1650 ofhuman IGF2R, wherein E1544 is substituted for a non-acidic residue, andwherein said binding domain binds IGF-II with increased affinityrelative to residues 1511 to 1650 of human IGF2R.
 2. A mutant IGF-IIbinding domain according to claim 1 which shows no binding orsubstantially no binding to IGF1.
 3. A mutant IGF-II binding domainaccording to claim 1 wherein the mutated residues are mutated bysubstitution, insertion or deletion.
 4. A mutant IGF-II binding domainaccording to claim 1 wherein residue E1544 is substituted for a basicresidue.
 5. A mutant IGF-II binding domain according to claim 4 whereinE1544 is substituted for K.
 6. A mutant IGF-II binding domain accordingto claim 4 wherein E1544 is substituted for R.
 7. A mutant IGF-IIbinding domain according to claim 4 wherein E1544 is substituted for H.8. A mutant IGF-II binding domain according to claim 4 wherein E1544 issubstituted for S.
 9. A mutant IGF-II binding domain according to claim1 wherein residues F1567 and I1572 are not mutated.
 10. A mutant IGF-IIbinding domain according to claim 9 wherein T1570 is not mutated.
 11. Amutant IGF-II binding domain according to claim 9 wherein P1597 andP1599 are not mutated.
 12. A mutant IGF-II binding domain according toclaim 1 which consists of the amino acid sequence of residues 1511 to1650 of human IGF2R with residue E1544 substituted for a non-hydrophobicresidue.
 13. A polypeptide comprising an IGF-II binding domain accordingto claim
 1. 14. A polypeptide according to claim 13 comprising two ormore mutant IGF-II binding domains.
 15. A polypeptide according to claim13 comprising domain 13 of human IGF2R.
 16. A polypeptide according toclaim 13 comprising an immunoglobulin Fc domain.
 17. A polypeptideaccording to claim 13 comprising an affinity tag.
 18. A nucleic acidencoding an IGF-II binding domain according to claim
 1. 19. A vectorcomprising a nucleic acid according to claim
 18. 20. A host cellcomprising a vector according to claim
 19. 21. A mutant IGF-II bindingdomain comprising the amino acid sequence of residues 1511 to 1650 ofhuman IGF2R with 5 or fewer of said residues mutated, wherein E1544 issubstituted for a non-acidic residue, and wherein said binding domainbinds IGF-II with increased affinity relative to residues 1511 to 1650of human IGF2R.